Chapter 13

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13
Bioenergetics and Biochemical
Reaction Types
13.1 Bioenergetics and Thermodynamics 506 candle, and that, from this point of view, animals
that respire are true combustible bodies that burn
13.2 Chemical Logic and Common Biochemical Reactions 511 and consume themselves. . . . One may say that this
13.3 Phosphoryl Group Transfers and ATP 517 analogy between combustion and respiration has
not escaped the notice of the poets, or rather the
13.4 Biological Oxidation-Reduction Reactions 528 philosophers of antiquity, and which they had
expounded and interpreted. This fire stolen from
L
iving cells and organisms must perform work to stay heaven, this torch of Prometheus, does not only
alive, to grow, and to reproduce. The ability to har- represent an ingenious and poetic idea, it is a faith-
ness energy and to channel it into biological work is ful picture of the operations of nature, at least for
a fundamental property of all living organisms; it must animals that breathe; one may therefore say, with
have been acquired very early in cellular evolution. the ancients, that the torch of life lights itself at the
Modern organisms carry out a remarkable variety of moment the infant breathes for the first time, and it
energy transductions, conversions of one form of energy does not extinguish itself except at death.*
to another. They use the chemical energy in fuels to In the twentieth century, we began to understand
bring about the synthesis of complex, highly ordered much of the chemistry underlying that “torch of life.”
macromolecules from simple precursors. They also con- Biological energy transductions obey the same chemical
vert the chemical energy of fuels into concentration and physical laws that govern all other natural processes.
gradients and electrical gradients, into motion and heat, It is therefore essential for a student of biochemistry to
and, in a few organisms such as fireflies and deep-sea understand these laws and how they apply to the flow
fish, into light. Photosynthetic organisms transduce of energy in the biosphere.
light energy into all these other forms of energy. In this chapter we first review the laws of thermody-
The chemical mechanisms that underlie biological namics and the quantitative relationships among free
energy transductions have fascinated and challenged energy, enthalpy, and entropy. We then review the com-
biologists for centuries. The mon types of biochemical reactions that occur in living
French chemist Antoine Lavo- cells, reactions that harness, store, transfer, and release
isier recognized that animals the energy taken up by organisms from their surround-
somehow transform chemical ings. Our focus then shifts to reactions that have special
fuels (foods) into heat and roles in biological energy exchanges, particularly those
that this process of respiration involving ATP. We finish by considering the importance of
is essential to life. He observed oxidation-reduction reactions in living cells, the energet-
that ics of biological electron transfers, and the electron carri-
. . . in general, respiration is ers commonly employed as cofactors in these processes.
nothing but a slow combustion
of carbon and hydrogen, which *From a memoir by Armand Seguin and Antoine Lavoisier, dated
Antoine Lavoisier, is entirely similar to that which 1789, quoted in Lavoisier, A. (1862) Oeuvres de Lavoisier,
1743–1794 occurs in a lighted lamp or Imprimerie Impériale, Paris.
505
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506 Bioenergetics and Biochemical Reaction Types
13.1 Bioenergetics and Thermodynamics it may be an organism, a cell, or two reacting compounds.
The reacting system and its surroundings together con-
Bioenergetics is the quantitative study of energy stitute the universe. In the laboratory, some chemical or
transductions—changes of one form of energy into physical processes can be carried out in isolated or
another—that occur in living cells, and of the nature closed systems, in which no material or energy is
and function of the chemical processes underlying these exchanged with the surroundings. Living cells and
transductions. Although many of the principles of ther- organisms, however, are open systems, exchanging both
modynamics have been introduced in earlier chapters material and energy with their surroundings; living sys-
and may be familiar to you, a review of the quantitative tems are never at equilibrium with their surroundings,
aspects of these principles is useful here. and the constant transactions between system and sur-
roundings explain how organisms can create order
Biological Energy Transformations Obey within themselves while operating within the second
law of thermodynamics.
the Laws of Thermodynamics In Chapter 1 (p. 23) we defined three thermody-
Many quantitative observations made by physicists and namic quantities that describe the energy changes
chemists on the interconversion of different forms of occurring in a chemical reaction:
energy led, in the nineteenth century, to the formulation
of two fundamental laws of thermodynamics. The first Gibbs free energy, G, expresses the amount of an
law is the principle of the conservation of energy: for energy capable of doing work during a reaction at
any physical or chemical change, the total amount of constant temperature and pressure. When a reac-
energy in the universe remains constant; energy tion proceeds with the release of free energy (that
may change form or it may be transported from one is, when the system changes so as to possess less
region to another, but it cannot be created or free energy), the free-energy change, DG, has a
destroyed. The second law of thermodynamics, which negative value and the reaction is said to be exer-
can be stated in several forms, says that the universe gonic. In endergonic reactions, the system gains
always tends toward increasing disorder: in all natural free energy and DG is positive.
processes, the entropy of the universe increases.
Enthalpy, H, is the heat content of the reacting
system. It reflects the number and kinds of chemi-
cal bonds in the reactants and products. When a
chemical reaction releases heat, it is said to be exo-
thermic; the heat content of the products is less
than that of the reactants and DH has, by conven-
tion, a negative value. Reacting systems that take
up heat from their surroundings are endothermic
and have positive values of DH.
Entropy, S, is a quantitative expression for the
randomness or disorder in a system (see Box 1–3).
When the products of a reaction are less complex
and more disordered than the reactants, the reac-
tion is said to proceed with a gain in entropy.
The units of DG and DH are joules/mole or calories/mole

(recall that 1 cal 5 4.184 J); units of entropy are joules/
mole ? Kelvin (J/mol ? K) (Table 13–1).
Under the conditions existing in biological systems
(including constant temperature and pressure), changes
Living organisms consist of collections of molecules in free energy, enthalpy, and entropy are related to each
much more highly organized than the surrounding other quantitatively by the equation
materials from which they are constructed, and organ-
¢G 5 ¢H 2 T ¢S (13–1)
isms maintain and produce order, seemingly immune to
the second law of thermodynamics. But living organisms in which DG is the change in Gibbs free energy of the
do not violate the second law; they operate strictly reacting system, DH is the change in enthalpy of the sys-
within it. To discuss the application of the second law to tem, T is the absolute temperature, and DS is the
biological systems, we must first define those systems change in entropy of the system. By convention, DS has
and their surroundings. a positive sign when entropy increases and DH, as noted
The reacting system is the collection of matter that above, has a negative sign when heat is released by the
is undergoing a particular chemical or physical process; system to its surroundings. Either of these conditions,
13.1 Bioenergetics and Thermodynamics 507
concentration of reactants and products, the rates of

TABLE 13–1 Some Physical Constants and Units the forward and reverse reactions are exactly equal and
Used in Thermodynamics no further net change occurs in the system. The con-
Boltzmann constant, k 5 1.381 3 10223 J/K centrations of reactants and products at equilibrium
Avogadro’s number, N 5 6.022 3 1023 mol21 define the equilibrium constant, Keq (p. 25). In the gen-
Faraday constant, 5 96,480 J/V?mol eral reaction aA 1 bB ∆ cC 1 dD, where a, b, c,
Gas constant, R 5 8.315 J/mol? K and d are the number of molecules of A, B, C, and D
(5 1.987 cal/mol? K) participating, the equilibrium constant is given by
Units of DG and DH are J/mol (or cal/mol) [C]c[D]d
!eq 5 (13–2)
Units of ¢S are J/mol? K (or cal/mol? K) [A]a[B]b
1 cal 5 4.184 J
where [A], [B], [C], and [D] are the molar concentrations
Units of absolute temperature, T, are Kelvin, K of the reaction components at the point of equilibrium.
25 8C 5 298 K When a reacting system is not at equilibrium, the
At 25 8C, RT 5 2.478 kJ/mol tendency to move toward equilibrium represents a
(5 0.592 kcal/mol) driving force, the magnitude of which can be expressed
as the free-energy change for the reaction, DG. Under
standard conditions (298 K 5 25 8C), when reactants
and products are initially present at 1 M concentrations
which are typical of energetically favorable processes,
or, for gases, at partial pressures of 101.3 kilopascals
tend to make DG negative. In fact, DG of a spontane-
(kPa), or 1 atm, the force driving the system toward
ously reacting system is always negative.
equilibrium is defined as the standard free-energy
The second law of thermodynamics states that the
change, DG8. By this definition, the standard state for
entropy of the universe increases during all chemical
reactions that involve hydrogen ions is [H1] 5 1 M, or
and physical processes, but it does not require that the
pH 0. Most biochemical reactions, however, occur in
entropy increase take place in the reacting system
well-buffered aqueous solutions near pH 7; both the pH
itself. The order produced within cells as they grow and
and the concentration of water (55.5 M) are essentially
divide is more than compensated for by the disorder
constant.
they create in their surroundings in the course of
growth and division (see Box 1–3, case 2). In short, liv- KEY CONVENTION: For convenience of calculations, bio-
ing organisms preserve their internal order by taking chemists define a standard state different from that
from the surroundings free energy in the form of nutri- used in chemistry and physics: in the biochemical
ents or sunlight, and returning to their surroundings an standard state, [H1] is 1027 M (pH 7) and [H2O] is 55.5 M.
equal amount of energy as heat and entropy. For reactions that involve Mg21 (which include most of
those with ATP as a reactant), [Mg21] in solution is com-
Cells Require Sources of Free Energy monly taken to be constant at 1 mM. ■
Cells are isothermal systems—they function at essentially
constant temperature (and also function at constant pres- Physical constants based on this biochemical stan-
sure). Heat flow is not a source of energy for cells, dard state are called standard transformed con-
because heat can do work only as it passes to a zone or stants and are written with a prime (such as DG98 and
object at a lower temperature. The energy that cells can K9eq) to distinguish them from the untransformed con-
and must use is free energy, described by the Gibbs free- stants used by chemists and physicists. (Note that most
energy function G, which allows prediction of the direc- other textbooks use the symbol DG89 rather than DG98.
tion of chemical reactions, their exact equilibrium posi- Our use of DG98, recommended by an international com-
tion, and the amount of work they can (in theory) perform mittee of chemists and biochemists, is intended to
at constant temperature and pressure. Heterotrophic emphasize that the transformed free energy, DG9, is the
cells acquire free energy from nutrient molecules, and criterion for equilibrium.) For simplicity, we will hereaf-
photosynthetic cells acquire it from absorbed solar radia- ter refer to these transformed constants as standard
tion. Both kinds of cells transform this free energy into free-energy changes.
ATP and other energy-rich compounds capable of provid-
ing energy for biological work at constant temperature. KEY CONVENTION: In another simplifying convention used
by biochemists, when H2O, H1, and/or Mg21 are reac-
tants or products, their concentrations are not included
Standard Free-Energy Change Is Directly Related
in equations such as Equation 13–2 but are instead
to the Equilibrium Constant incorporated into the constants K¿eq and DG98. ■
The composition of a reacting system (a mixture of
chemical reactants and products) tends to continue Just as K¿eq is a physical constant characteristic for
changing until equilibrium is reached. At the equilibrium each reaction, so too is DG98 a constant. As we noted in
Chapter 6, there is a simple relationship between K¿eq

and DG98: TABLE 13–3 Relationships among K9eq, DG98, and
the Direction of Chemical Reactions
¢G¿8 5 2R" ln K¿eq (13–3)
Starting with all
The standard free-energy change of a chemical components at 1 M,
reaction is simply an alternative mathematical way When K9eq is . . . DG98 is . . . the reaction . . .
of expressing its equilibrium constant. Table 13–2
shows the relationship between DG98 and K¿eq. If the .1.0 negative proceeds forward
equilibrium constant for a given chemical reaction is 1.0 zero is at equilibrium
1.0, the standard free-energy change of that reaction ,1.0 positive proceeds in reverse
is 0.0 (the natural logarithm of 1.0 is zero). If K¿eq of a
reaction is greater than 1.0, its DG98 is negative. If K¿eq
is less than 1.0, DG98 is positive. Because the relation-
ship between DG98 and K¿eq is exponential, relatively
small changes in DG98 correspond to large changes
WORKED EXAMPLE 13–1 Calculation of DG98
in K¿eq. Calculate the standard free-energy change of the reac-
It may be helpful to think of the standard free- tion catalyzed by the enzyme phosphoglucomutase
energy change in another way. ¢G¿8 is the difference
Glucose 1-phosphate ∆ glucose 6-phosphate
between the free-energy content of the products and
the free-energy content of the reactants, under stan- given that, starting with 20 mM glucose 1-phosphate and
dard conditions. When DG98 is negative, the products no glucose 6-phosphate, the final equilibrium mixture at
contain less free energy than the reactants and the 25 8C and pH 7.0 contains 1.0 mM glucose 1-phosphate
reaction will proceed spontaneously under standard and 19 mM glucose 6-phosphate. Does the reaction in
conditions; all chemical reactions tend to go in the the direction of glucose 6-phosphate formation proceed
direction that results in a decrease in the free energy of with a loss or a gain of free energy?
the system. A positive value of DG98 means that the
products of the reaction contain more free energy than Solution: First we calculate the equilibrium constant:
the reactants, and this reaction will tend to go in the [glucose 6-phosphate 4 19 mM
reverse direction if we start with 1.0 M concentrations of K¿eq 5 5 5 19
[glucose 1-phosphate 4 1.0 mM
all components (standard conditions). Table 13–3 sum-
marizes these points. We can now calculate the standard free-energy change:
¢G¿8 5 2RT ln K¿eq
5 2(8.315 J/mol?K)(298 K)(ln 19)
5 27.3 kJ/mol
TABLE 13–2 Relationship between Equilibrium Because the standard free-energy change is negative,
Constants and Standard Free-Energy the conversion of glucose 1-phosphate to glucose
Changes of Chemical Reactions 6-phosphate proceeds with a loss (release) of free energy.
(For the reverse reaction, DG98 has the same magnitude
DG98 but the opposite sign.)
K9eq (kJ/mol) (kcal/mol)*
103 217.1 24.1
Table 13–4 gives the standard free-energy changes
102 211.4 22.7
for some representative chemical reactions. Note that
101 25.7 21.4 hydrolysis of simple esters, amides, peptides, and glyco-
1 0.0 0.0 sides, as well as rearrangements and eliminations, pro-
1021 5.7 1.4 ceed with relatively small standard free-energy changes,
whereas hydrolysis of acid anhydrides is accompanied
1022 11.4 2.7
by relatively large decreases in standard free energy.
1023 17.1 4.1 The complete oxidation of organic compounds such as
1024 22.8 5.5 glucose or palmitate to CO2 and H2O, which in cells
10 25
28.5 6.8 requires many steps, results in very large decreases in
standard free energy. However, standard free-energy
1026 34.2 8.2
changes such as those in Table 13–4 indicate how much
*Although joules and kilojoules are the standard units of energy and are used throughout free energy is available from a reaction under standard
this text, biochemists and nutritionists sometimes express DG98 values in kilocalories per conditions. To describe the energy released under the
mole. We have therefore included values in both kilojoules and kilocalories in this table and
in Tables 13–4 and 13–6. To convert kilojoules to kilocalories, divide the number of conditions existing in cells, an expression for the actual
kilojoules by 4.184. free-energy change is essential.
13.1 Bioenergetics and Thermodynamics 509
TABLE 13–4 Standard Free-Energy Changes of Some Chemical Reactions

DG98
Reaction type (kJ/mol) (kcal/mol)
Hydrolysis reactions
Acid anhydrides
Acetic anhydride 1 H2O 88n 2 acetate 291.1 221.8
ATP 1 H2O 88n ADP 1 Pi 230.5 27.3
ATP 1 H2O 88n AMP 1 PPi 245.6 210.9
PPi 1 H2O 88n 2Pi 219.2 24.6
UDP-glucose 1 H2O 88n UMP 1 glucose 1-phosphate 243.0 210.3
Esters
Ethyl acetate 1 H2O 88n ethanol 1 acetate 219.6 24.7
Glucose 6-phosphate 1 H2O 88n glucose 1 Pi 213.8 23.3
Amides and peptides
Glutamine 1 H2O 88n glutamate 1 NH 14 214.2 23.4
Glycylglycine 1 H2O 88n 2 glycine 29.2 22.2
Glycosides
Maltose 1 H2O 88n 2 glucose 215.5 23.7
Lactose 1 H2O 88n glucose 1 galactose 215.9 23.8
Rearrangements
Glucose 1-phosphate 88n glucose 6-phosphate 27.3 21.7
Fructose 6-phosphate 88n glucose 6-phosphate 21.7 20.4
Elimination of water
Malate 88n fumarate 1 H2O 3.1 0.8
Oxidations with molecular oxygen
Glucose 1 6O2 88n 6CO2 1 6H2O 22,840 2686
Palmitate 1 23O2 88n 16CO2 1 16H2O 29,770 22,338
Actual Free-Energy Changes Depend on Reactant will necessarily match the standard conditions as defined
above. Moreover, the DG of any reaction proceeding
and Product Concentrations spontaneously toward its equilibrium is always negative,
We must be careful to distinguish between two different becomes less negative as the reaction proceeds, and is
quantities: the actual free-energy change, DG, and the zero at the point of equilibrium, indicating that no more
standard free-energy change, DG98. Each chemical reac- work can be done by the reaction.
tion has a characteristic standard free-energy change, DG and DG98 for any reaction aA 1 bB ∆ cC 1 dD
which may be positive, negative, or zero, depending on are related by the equation
the equilibrium constant of the reaction. The standard
free-energy change tells us in which direction and how [C]c[D]d
far a given reaction must go to reach equilibrium when ¢G 5 ¢G¿8 1 RT ln (13–4)
[A]a[B]b
the initial concentration of each component is 1.0 M,
the pH is 7.0, the temperature is 25 8C, and the pressure in which the terms in red are those actually prevailing
is 101.3 kPa (1 atm). Thus DG98 is a constant: it has a in the system under observation. The concentration
characteristic, unchanging value for a given reaction. terms in this equation express the effects commonly
But the actual free-energy change, DG, is a function of called mass action, and the term [C]c[D]d/[A]a[B]b is called
reactant and product concentrations and of the tem- the mass-action ratio, Q. Thus Equation 13–4 can be
perature prevailing during the reaction, none of which expressed as DG 5 DG98 1 RT ln Q. As an example, let
us suppose that the reaction A 1 B ∆ C 1 D is tak- supplying additional heat but by lowering the activation
ing place under the standard conditions of temperature energy through use of an enzyme. An enzyme provides
(25 8C) and pressure (101.3 kPa) but that the concen- an alternative reaction pathway with a lower activation
trations of A, B, C, and D are not equal and none of the energy than the uncatalyzed reaction, so that at room
components is present at the standard concentration of temperature a large fraction of the substrate molecules
1.0 M. To determine the actual free-energy change, DG, have enough thermal energy to overcome the activation
under these nonstandard conditions of concentration barrier, and the reaction rate increases dramatically.
as the reaction proceeds from left to right, we simply The free-energy change for a reaction is indepen-
enter the actual concentrations of A, B, C, and D in dent of the pathway by which the reaction occurs; it
Equation 13–4; the values of R, T, and DG98 are the depends only on the nature and concentration of the
standard values. DG is negative and approaches zero as initial reactants and the final products. Enzymes can-
the reaction proceeds, because the actual concentrations not, therefore, change equilibrium constants; but
of A and B decrease and the concentrations of C and D they can and do increase the rate at which a reaction
increase. Notice that when a reaction is at equilibrium— proceeds in the direction dictated by thermodynamics
when there is no force driving the reaction in either (see Section 6.2).
direction and ¢G is zero—Equation 13–4 reduces to
0 5 ¢G 5 ¢G¿8 1 RT ln
[C]eq[D]eq
Standard Free-Energy Changes Are Additive
[A]eq[B]eq
In the case of two sequential chemical reactions,
or A ∆ B and B ∆ C, each reaction has its own
equilibrium constant and each has its characteristic
¢G¿8 5 2RT ln K¿eq
standard free-energy change, ¢G¿18 and ¢G¿28. As the
which is the equation relating the standard free-energy two reactions are sequential, B cancels out to give the
change and equilibrium constant (Eqn 13–3). overall reaction A ∆ C, which has its own equilibrium
The criterion for spontaneity of a reaction is the constant and thus its own standard free-energy
value of DG, not DG98. A reaction with a positive DG98 can change, ¢G¿8 total. The DG98 values of sequential chemi-
go in the forward direction if DG is negative. This is pos- cal reactions are additive. For the overall reaction
sible if the term RT ln ([products]/[reactants]) in Equa- A ∆ C, ¢G¿8 total is the sum of the individual standard
tion 13–4 is negative and has a larger absolute value than free-energy changes, ¢G¿18 and ¢G¿28, of the two reac-
DG98. For example, the immediate removal of the prod- tions: ¢G¿8
total 5 ¢G¿18 1 ¢G¿28.
ucts of a reaction can keep the ratio [products]/[reactants]
(1) A 88n B ¢G¿18
well below 1, such that the term RT ln ([products]/
(2) B 88n C ¢G¿28
[reactants]) has a large, negative value. DG98 and DG are
expressions of the maximum amount of free energy that Sum: A 88n C ¢G¿18 1 ¢G¿8
2
a given reaction can theoretically deliver—an amount of This principle of bioenergetics explains how a thermo-
energy that could be realized only if a perfectly efficient dynamically unfavorable (endergonic) reaction can be
device were available to trap or harness it. Given that no driven in the forward direction by coupling it to a
such device is possible (some energy is always lost to highly exergonic reaction through a common interme-
entropy during any process), the amount of work done diate. For example, the synthesis of glucose 6-phos-
by the reaction at constant temperature and pressure is phate is the first step in the utilization of glucose by
always less than the theoretical amount. many organisms:
Another important point is that some thermody-
namically favorable reactions (that is, reactions for Glucose 1 Pi 88n glucose 6-phosphate 1 H2O
which DG98 is large and negative) do not occur at mea- ¢G¿8 5 13.8 kJ/mol
surable rates. For example, combustion of firewood to The positive value of DG98 predicts that under standard
CO2 and H2O is very favorable thermodynamically, but conditions the reaction will tend not to proceed spon-
firewood remains stable for years because the activation taneously in the direction written. Another cellular
energy (see Figs 6–2 and 6–3) for the combustion reac- reaction, the hydrolysis of ATP to ADP and Pi, is very
tion is higher than the energy available at room tem- exergonic:
perature. If the necessary activation energy is provided
(with a lighted match, for example), combustion will ATP 1 H2O 88n ADP 1 Pi ¢G¿8 5 230.5 kJ/mol
begin, converting the wood to the more stable products These two reactions share the common intermediates
CO2 and H2O and releasing energy as heat and light. The Pi and H2O and may be expressed as sequential
heat released by this exothermic reaction provides the reactions:
activation energy for combustion of neighboring regions
of the firewood; the process is self-perpetuating. (1) Glucose 1 Pi 88n glucose 6-phosphate 1 H2O
In living cells, reactions that would be extremely (2) ATP 1 H2O 88n ADP 1 Pi
slow if uncatalyzed are caused to proceed not by Sum: ATP 1 glucose 88n ADP 1 glucose 6-phosphate
13.2 Chemical Logic and Common Biochemical Reactions 511
The overall standard free-energy change is obtained by ! Bioenergetics is the quantitative study of energy
adding the DG98 values for individual reactions: relationships and energy conversions in biological
systems. Biological energy transformations obey
¢G¿8 5 13.8 kJ/mol 1 (230.5 kJ/mol) 5 216.7 kJ/mol
the laws of thermodynamics.
The overall reaction is exergonic. In this case, energy ! All chemical reactions are influenced by two
stored in ATP is used to drive the synthesis of glucose forces: the tendency to achieve the most stable
6-phosphate, even though its formation from glucose bonding state (for which enthalpy, H, is a useful
and inorganic phosphate (Pi) is endergonic. The path- expression) and the tendency to achieve the
way of glucose 6-phosphate formation from glucose by highest degree of randomness, expressed as
phosphoryl transfer from ATP is different from reac- entropy, S. The net driving force in a reaction is
tions (1) and (2) above, but the net result is the same DG, the free-energy change, which represents the
as the sum of the two reactions. In thermodynamic cal- net effect of these two factors: ¢G 5 ¢H 2 T ¢S.
culations, all that matters is the state of the system at
! The standard transformed free-energy change,
the beginning of the process and its state at the end; the
¢G¿8, is a physical constant that is characteristic
route between the initial and final states is immaterial.
for a given reaction and can be calculated from the
We have said that DG98 is a way of expressing the
equilibrium constant for the reaction:
equilibrium constant for a reaction. For reaction (1)
¢G¿8 5 2RT ln K¿eq.
above,
! The actual free-energy change, DG, is a
[glucose 6-phosphate ] variable that depends on DG98 and on the
!¿eq 1 5 5 3.9 3 1023 M21
[glucose ] [Pi ] concentrations of reactants and products:
Notice that H2O is not included in this expression, as its DG 5 DG98 1 RT ln ([products]/[reactants]).
concentration (55.5 M) is assumed to remain unchanged ! When DG is large and negative, the reaction tends
by the reaction. The equilibrium constant for the hy- to go in the forward direction; when DG is large
drolysis of ATP is and positive, the reaction tends to go in the
reverse direction; and when DG 5 0, the system is
[ADP] [Pi ]
!¿eq 2 5 5 2.0 3 105 M at equilibrium.
[ATP]
! The free-energy change for a reaction is
The equilibrium constant for the two coupled reactions is independent of the pathway by which the reaction
[glucose 6-phosphate ] [ADP] [Pi ] occurs. Free-energy changes are additive; the net
!¿eq 3 5
[glucose ] [Pi ] [ATP]
chemical reaction that results from successive
reactions sharing a common intermediate has an
5 (!¿eq1 ) (K¿eq2 ) 5 (3.9 3 1023 M21 ) (2.0 3 105 M )
overall free-energy change that is the sum of the
5 7.8 3 102
DG values for the individual reactions.
This calculation illustrates an important point about
equilibrium constants: although the DG98 values for two
reactions that sum to a third, overall reaction are addi- 13.2 Chemical Logic and Common
tive, the K¿eq for the overall reaction is the product of
the individual K¿eq values for the two reactions. Equilib- Biochemical Reactions
rium constants are multiplicative. By coupling ATP The biological energy transductions we are concerned
hydrolysis to glucose 6-phosphate synthesis, the K¿eq for with in this book are chemical reactions. Cellular chem-
formation of glucose 6-phosphate from glucose has istry does not encompass every kind of reaction learned
been raised by a factor of about 2 3 105. in a typical organic chemistry course. Which reactions
This common-intermediate strategy is employed by take place in biological systems and which do not is
all living cells in the synthesis of metabolic intermediates determined by (1) their relevance to that particular
and cellular components. Obviously, the strategy works metabolic system and (2) their rates. Both consider-
only if compounds such as ATP are continuously avail- ations play major roles in shaping the metabolic path-
able. In the following chapters we consider several of the ways we consider throughout the rest of the book. A
most important cellular pathways for producing ATP. relevant reaction is one that makes use of an available
substrate and converts it to a useful product. However,
even a potentially relevant reaction may not occur.
SUMMARY 13.1 Bioenergetics and Thermodynamics Some chemical transformations are too slow (have acti-
! Living cells constantly perform work. They require vation energies that are too high) to contribute to living
energy for maintaining their highly organized systems even with the aid of powerful enzyme catalysts.
structures, synthesizing cellular components, The reactions that do occur in cells represent a toolbox
generating electric currents, and many other that evolution has used to construct metabolic pathways
processes. that circumvent the “impossible” reactions. Learning to
recognize the plausible reactions can be a great aid in Nucleophiles Electrophiles

developing a command of biochemistry.
:R
Even so, the number of metabolic transformations
O–: C
taking place in a typical cell can seem overwhelming.
Negatively charged O
Most cells have the capacity to carry out thousands of oxygen (as in an
specific, enzyme-catalyzed reactions: for example, unprotonated hydroxyl Carbon atom of a
group or an ionized carbonyl group (the
transformation of a simple nutrient such as glucose into more electronegative
carboxylic acid)
amino acids, nucleotides, or lipids; extraction of energy oxygen of the carbonyl
group pulls electrons
from fuels by oxidation; and polymerization of mono- S–
:
away from the carbon)
meric subunits into macromolecules. Negatively charged
To study these reactions, some organization is essen- sulfhydryl :R
+
tial. There are patterns within the chemistry of life; you do C N
not need to learn every individual reaction to comprehend C–:
H
the molecular logic of biochemistry. Most of the reactions Carbanion Protonated imine group
in living cells fall into one of five general categories: (activated for nucleophilic
(1) reactions that make or break carbon–carbon bonds; attack at the carbon by
:
N
protonation of the imine)
(2) internal rearrangements, isomerizations, and elimina-
Uncharged
tions; (3) free-radical reactions; (4) group transfers; and amine group O– :R
(5) oxidation-reductions. We discuss each of these in –O
P O
more detail below and refer to some examples of each
O–
type in later chapters. Note that the five reaction types HN N:
Phosphorus of
are not mutually exclusive; for example, an isomerization a phosphate group
Imidazole
reaction may involve a free-radical intermediate. :R
Before proceeding, however, we should review two H O–: H+
basic chemical principles. First, a covalent bond con-
Hydroxide ion Proton
sists of a shared pair of electrons, and the bond can be
broken in two general ways (Fig. 13–1). In homolytic FIGURE 13–2 Common nucleophiles and electrophiles in biochemical
cleavage, each atom leaves the bond as a radical, reactions. Chemical reaction mechanisms, which trace the formation
carrying one unpaired electron. In heterolytic cleavage, and breakage of covalent bonds, are communicated with dots and
curved arrows, a convention known informally as “electron pushing.” A
covalent bond consists of a shared pair of electrons. Nonbonded elec-
Homolytic . . trons important to the reaction mechanism are designated by dots (:).
C H C ! H
cleavage
Curved arrows ( ) represent the movement of electron pairs. For
Carbon H atom movement of a single electron (as in a free radical reaction), a single-
radical headed (fishhook-type) arrow is used ( ). Most reaction steps
involve an unshared electron pair.
. .
C C C ! C
which is more common, one atom retains both bonding
Carbon radicals
electrons. The species most often generated when COC
and COH bonds are cleaved are illustrated in Figure
Heterolytic 13–1. Carbanions, carbocations, and hydride ions are
C H C :– ! H+
cleavage highly unstable; this instability shapes the chemistry of
Carbanion Proton
these ions, as we shall see.
The second basic principle is that many biochemical
reactions involve interactions between nucleophiles
C H C+ ! H:– (functional groups rich in and capable of donating elec-
trons) and electrophiles (electron-deficient functional
Carbocation Hydride groups that seek electrons). Nucleophiles combine with
and give up electrons to electrophiles. Common biological
nucleophiles and electrophiles are shown in Figure 13–2.
C C C :– ! +C Note that a carbon atom can act as either a nucleophile
or an electrophile, depending on which bonds and func-
Carbanion Carbocation tional groups surround it.
FIGURE 13–1 Two mechanisms for cleavage of a COC or COH bond.
In a homolytic cleavage, each atom keeps one of the bonding electrons, Reactions That Make or Break Carbon–Carbon Bonds Hetero-
resulting in the formation of carbon radicals (carbons having unpaired lytic cleavage of a COC bond yields a carbanion and a
electrons) or uncharged hydrogen atoms. In a heterolytic cleavage, one carbocation (Fig. 13–1). Conversely, the formation of a
of the atoms retains both bonding electrons. This can result in the for- COC bond involves the combination of a nucleophilic
mation of carbanions, carbocations, protons, or hydride ions. carbanion and an electrophilic carbocation. Carbanions
and carbocations are generally so unstable that their O R2 R3 O R2 R3

H!
formation as reaction intermediates can be energetically R1 C C "
C O R1 C C C OH
inaccessible even with enzyme catalysts. For the purpose
H R4 H R4
of cellular biochemistry they are impossible reactions—
Aldol condensation
unless chemical assistance is provided in the form of
functional groups containing electronegative atoms
O H R1 O H R1
(O and N) that can alter the electronic structure of adja- H! HS — R2
cent carbon atoms so as to stabilize and facilitate the CoA-S C C "
C O CoA-S C C C O
formation of carbanion and carbocation intermediates. H S — R2 H
Carbonyl groups are particularly important in the Claisen ester condensation
chemical transformations of metabolic pathways.
The carbon of a carbonyl group has a partial positive O H O H
O H!
charge due to the electron-withdrawing property of the
R C C C R C C H ! CO2
carbonyl oxygen, and thus is an electrophilic carbon
H O "
H
(Fig. 13–3a). A carbonyl group can thus facilitate the
formation of a carbanion on an adjoining carbon by delo- Decarboxylation of a b-keto acid
calizing the carbanion’s negative charge (Fig. 13–3b). FIGURE 13–4 Some common reactions that form and break COC
An imine group (see Fig. 1–16) can serve a similar func- bonds in biological systems. For both the aldol condensation and the
tion (Fig. 13–3c). The capacity of carbonyl and imine Claisen condensation, a carbanion serves as nucleophile and the carbon
groups to delocalize electrons can be further enhanced of a carbonyl group serves as electrophile. The carbanion is stabilized in
by a general acid catalyst or by a metal ion such as Mg21 each case by another carbonyl at the adjoining carbon. In the decarbox-
(Fig. 13–3d). ylation reaction, a carbanion is formed on the carbon shaded blue as the
The importance of a carbonyl group is evident in CO2 leaves. The reaction would not occur at an appreciable rate without
three major classes of reactions in which COC bonds the stabilizing effect of the carbonyl adjacent to the carbanion carbon.
are formed or broken (Fig. 13–4): aldol condensations, Wherever a carbanion is shown, a stabilizing resonance with the adjacent
Claisen ester condensations, and decarboxylations. In carbonyl, as shown in Figure 13–3b, is assumed. An imine (Fig. 13–3c) or
each type of reaction, a carbanion intermediate is sta- other electron-withdrawing group (including certain enzymatic cofactors
bilized by a carbonyl group, and in many cases another such as pyridoxal) can replace the carbonyl group in the stabilization of
carbonyl provides the electrophile with which the carbanions.
nucleophilic carbanion reacts.
An aldol condensation is a common route to the citric acid cycle (see Fig. 16–9). Decarboxylation also
formation of a COC bond; the aldolase reaction, which commonly involves the formation of a carbanion stabi-
converts a six-carbon compound to two three-carbon lized by a carbonyl group; the acetoacetate decarboxyl-
compounds in glycolysis, is an aldol condensation in ase reaction that occurs in the formation of ketone bodies
reverse (see Fig. 14–6). In a Claisen condensation, during fatty acid catabolism provides an example (see
the carbanion is stabilized by the carbonyl of an adjacent Fig. 17–19). Entire metabolic pathways are organized
thioester; an example is the synthesis of citrate in the around the introduction of a carbonyl group in a particu-
lar location so that a nearby carbon–carbon bond can be
formed or cleaved. In some reactions, an imine or a spe-
O"" O O" cialized cofactor such as pyridoxal phosphate plays the
(a) C "!
(b) C C
"
mn C C electron-withdrawing role of the carbonyl group.
The carbocation intermediate occurring in some
!
NH2 NH2 reactions that form or cleave COC bonds is generated by
the elimination of a very good leaving group, such as pyro-
(c) C C C" mn C C C
phosphate (see Group Transfer Reactions below). An
example is the prenyltransferase reaction (Fig. 13–5), an
Me2! HA early step in the pathway of cholesterol biosynthesis.
ø
O O
(d) C C Internal Rearrangements, Isomerizations, and Eliminations
FIGURE 13–3 Chemical properties of carbonyl groups. (a) The carbon Another common type of cellular reaction is an intramo-
atom of a carbonyl group is an electrophile by virtue of the electron-
lecular rearrangement in which redistribution of electrons
withdrawing capacity of the electronegative oxygen atom, which results in alterations of many different types without a
results in a structure in which the carbon has a partial positive charge. change in the overall oxidation state of the molecule. For
(b) Within a molecule, delocalization of electrons into a carbonyl group example, different groups in a molecule may undergo
stabilizes a carbanion on an adjacent carbon, facilitating its formation. oxidation-reduction, with no net change in oxidation
(c) Imines function much like carbonyl groups in facilitating electron state of the molecule; groups at a double bond may
withdrawal. (d) Carbonyl groups do not always function alone; their undergo a cis-trans rearrangement; or the positions of
capacity as electron sinks often is augmented by interaction with either double bonds may be transposed. An example of an isom-
a metal ion (Me21, such as Mg21) or a general acid (HA). erization entailing oxidation-reduction is the formation of
CH3 fructose 6-phosphate from glucose 6-phosphate in gly-

O O H2 colysis (Fig. 13–6); this reaction is discussed in detail in
C C
J
G G
Chapter 14): C-1 is reduced (aldehyde to alcohol) and
G
"
O P O P O C CH3
H C-2 is oxidized (alcohol to ketone). Figure 13–6b shows
O" O"
the details of the electron movements in this type of
Dimethylallyl pyrophosphate
isomerization. A cis-trans rearrangement is illustrated by
the prolyl cis-trans isomerase reaction in the folding of
Isopentenyl PPi certain proteins (see Fig. 4–8). A simple transposition of
pyrophosphate
a CPC bond occurs during metabolism of oleic acid, a
common fatty acid (see Fig. 17–10). Some spectacular
CH3 examples of double-bond repositioning occur in the bio-
CH3 H2 synthesis of cholesterol (see Fig. 21–33).
!
C C
H2 J
G G
O O C G C C CH3 An example of an elimination reaction that does not
G
H affect overall oxidation state is the loss of water from an

G
"
O P O P O CG CH2
alcohol, resulting in the introduction of a CPC bond:
G
O" O" H H
Isopentenyl pyrophosphate Dimethylallylic carbocation H H H2O R H
R C C R1 C C
H! H2O H R1
H OH
O O Similar reactions can result from eliminations in amines.

"
O P O P O
Free-Radical Reactions Once thought to be rare, the homo-
O" O" lytic cleavage of covalent bonds to generate free radicals
Geranyl pyrophosphate has now been found in a wide range of biochemical
FIGURE 13–5 Carbocations in carbon–carbon bond formation. In one of processes. These include: isomerizations that make use
the early steps in cholesterol biosynthesis, the enzyme prenyltransferase of adenosylcobalamin (vitamin B12) or S-adenosyl-
catalyzes condensation of isopentenyl pyrophosphate and dimethylallyl methionine, which are initiated with a 59-deoxyadenosyl
pyrophosphate to form geranyl pyrophosphate (see Fig. 21–36). The radical (see the methylmalonyl-CoA mutase reaction in
reaction is initiated by elimination of pyrophosphate from the dimethyl- Box 17–2); certain radical-initiated decarboxylation
allyl pyrophosphate to generate a carbocation, stabilized by resonance reactions (Fig. 13–7); some reductase reactions, such as
with the adjacent CPC bond. that catalyzed by ribonucleotide reductase (see Fig. 22–41);
(a) H OH H H H O" H OH H H H O"

1 2 1 2
H C C C C C C O P O" H C C C C C C O P O"
phosphohexose
O OH H OH OH H O isomerase OH O H OH OH H O
Glucose 6-phosphate Fructose 6-phosphate
(b)
B1 : 1 B1 abstracts a B1 B1:
proton.
H 5 An electron pair is displaced
from the C C bond to form H
H 2 This allows the
C C
a C H bond with
formation of a C C the proton donated C C
C C double bond. by B1.
O O OH O
O OH H H
3 Electrons from 4 B2 abstracts a
H carbonyl form an proton, allowing
B2 : H
O H bond with the formation of
B2 the hydrogen ion aC O bond.
B2
donated by B2.
Enediol intermediate
FIGURE 13–6 Isomerization and elimination reactions. (a) The conver- follow the path of oxidation from left to right. B1 and B2 are ionizable
sion of glucose 6-phosphate to fructose 6-phosphate, a reaction of groups on the enzyme; they are capable of donating and accepting pro-
sugar metabolism catalyzed by phosphohexose isomerase. (b) This tons (acting as general acids or general bases) as the reaction proceeds.
reaction proceeds through an enediol intermediate. Light red screens
"
OOC "
OOC
•
X ! H •
e"
H3C R "XH ! H3C R H3C R
NH NH CO2 NH
R R R
Coproporphyrinogen III Coproporphyrinogenyl Protoporphyrinogen IX
III radical
FIGURE 13–7 A free radical–initiated decarboxylation reaction. The bio- decarboxylation via the free-radical mechanism shown here. The acceptor
synthesis of heme (see Fig. 22–26) in Escherichia coli includes a decarbox- of the released electron is not known. For simplicity, only the relevant
ylation step in which propionyl side chains on the coproporphyrinogen III portions of the large coproporphyrinogen III and protoporphyrinogen
intermediate are converted to the vinyl side chains of protoporphyrinogen IX. molecules are shown; the entire structures are given in Figure 22–26.
When the bacteria are grown anaerobically the enzyme oxygen-independent When E. coli are grown in the presence of oxygen, this reaction is an
coproporphyrinogen III oxidase, also called HemN protein, promotes oxidative decarboxylation and is catalyzed by a different enzyme.
and some rearrangement reactions, such as that cata- (a)

lyzed by DNA photolyase (see Fig. 25–26). O" O
"O P O "O
P O"
Group Transfer Reactions The transfer of acyl, glycosyl, and
O" O"
phosphoryl groups from one nucleophile to another is
common in living cells. Acyl group transfer generally
involves the addition of a nucleophile to the carbonyl car- O" O"
bon of an acyl group to form a tetrahedral intermediate: "O
P O" O P O"
O O"
O O" O (b)
C R C X C 3" O
O
R X Y R Y
:Y : X" O P O
Tetrahedral
O P
intermediate O
O O
The chymotrypsin reaction is one example of acyl group
transfer (see Fig. 6–22). Glycosyl group transfers (c) O O O
involve nucleophilic substitution at C-1 of a sugar ring, Adenine Ribose O P O P O P O" HO R
which is the central atom of an acetal. In principle, the Glucose
O" O" O"
substitution could proceed by an SN1 or SN2 pathway, as
ATP
described in Figure 6–28 for the enzyme lysozyme.
Phosphoryl group transfers play a special role in O O O
metabolic pathways, and these transfer reactions are
Adenine Ribose O P O P O" ! "
O P O R
discussed in detail in Section 13.3. A general theme in
metabolism is the attachment of a good leaving group to O" O" O "
a metabolic intermediate to “activate” the intermediate ADP Glucose 6-phosphate,

a phosphate ester
for subsequent reaction. Among the better leaving (d)
groups in nucleophilic substitution reactions are inor-
O O
ganic orthophosphate (the ionized form of H3PO4 at
22 Z P W Z#R OH
neutral pH, a mixture of H2PO2 4 and HPO4 , commonly
W # ADP
abbreviated Pi) and inorganic pyrophosphate (P2O42 7 ,
O
abbreviated PPi); esters and anhydrides of phosphoric FIGURE 13–8 Phosphoryl group transfers: some of the participants.
acid are effectively activated for reaction. Nucleophilic (a) In one (inadequate) representation of Pi, three oxygens are single-
substitution is made more favorable by the attachment bonded to phosphorus, and the fourth is double-bonded, allowing the
of a phosphoryl group to an otherwise poor leaving four different resonance structures shown here. (b) The resonance
group such as OOH. Nucleophilic substitutions in structures of Pi can be represented more accurately by showing all four
which the phosphoryl group (OPO22 3 ) serves as a leav- phosphorus–oxygen bonds with some double-bond character; the hybrid
ing group occur in hundreds of metabolic reactions. orbitals so represented are arranged in a tetrahedron with P at its center.
Phosphorus can form five covalent bonds. The (c) When a nucleophile Z (in this case, the OOH on C-6 of glucose)
conventional representation of Pi (Fig. 13–8a), with attacks ATP, it displaces ADP (W). In this SN2 reaction, a pentacovalent
three POO bonds and one PPO bond, is a convenient intermediate (d) forms transiently.
but inaccurate picture. In Pi, four equivalent phospho- OH 2H! ! 2e" O

O O
rus–oxygen bonds share some double-bond character,
CH3 CH C CH3 C C
and the anion has a tetrahedral structure (Fig. 13–8b). O" O"
Because oxygen is more electronegative than phos- 2H! ! 2e"
phorus, the sharing of electrons is unequal: the cen- Lactate lactate Pyruvate
dehydrogenase
tral phosphorus bears a partial positive charge and
can therefore act as an electrophile. In a great many FIGURE 13–10 An oxidation-reduction reaction. Shown here is the oxi-
metabolic reactions, a phosphoryl group (OPO22 3 ) is dation of lactate to pyruvate. In this dehydrogenation, two electrons and
transferred from ATP to an alcohol, forming a phos- two hydrogen ions (the equivalent of two hydrogen atoms) are removed
phate ester (Fig. 13–8c), or to a carboxylic acid, form- from C-2 of lactate, an alcohol, to form pyruvate, a ketone. In cells the
ing a mixed anhydride. When a nucleophile attacks reaction is catalyzed by lactate dehydrogenase and the electrons are
the electrophilic phosphorus atom in ATP, a relatively transferred to the cofactor nicotinamide adenine dinucleotide (NAD).
stable pentacovalent structure forms as a reaction This reaction is fully reversible; pyruvate can be reduced by electrons
intermediate (Fig. 13–8d). With departure of the leav- transferred from the cofactor.
ing group (ADP), the transfer of a phosphoryl group is
complete. The large family of enzymes that catalyze
phosphoryl group transfers with ATP as donor are
called kinases (Greek kinein, “to move”). Hexoki- compound loses two electrons and two hydrogen
nase, for example, “moves” a phosphoryl group from ions (that is, two hydrogen atoms); these reactions
ATP to glucose. are commonly called dehydrogenations and the
Phosphoryl groups are not the only groups that enzymes that catalyze them are called dehydrogenases
activate molecules for reaction. Thioalcohols (thiols), (Fig. 13–10). In some, but not all, biological oxida-
in which the oxygen atom of an alcohol is replaced tions, a carbon atom becomes covalently bonded to an
with a sulfur atom, are also good leaving groups. Thi- oxygen atom. The enzymes that catalyze these oxida-
ols activate carboxylic acids by forming thioesters tions are generally called oxidases or, if the oxygen
(thiol esters). In later chapters we discuss several atom is derived directly from molecular oxygen (O2)
reactions, including those catalyzed by the fatty acyl oxygenases.
synthases in lipid synthesis (see Fig. 21–2), in which Every oxidation must be accompanied by a reduc-
nucleophilic substitution at the carbonyl carbon of a tion, in which an electron acceptor acquires the elec-
thioester results in transfer of the acyl group to another trons removed by oxidation. Oxidation reactions gener-
moiety. ally release energy (think of camp fires: the compounds
in wood are oxidized by oxygen molecules in the air).
Oxidation-Reduction Reactions Carbon atoms can exist in Most living cells obtain the energy needed for cellular
five oxidation states, depending on the elements with work by oxidizing metabolic fuels such as carbohydrates
which they share electrons (Fig. 13–9), and transi- or fat (photosynthetic organisms can also trap and use
tions between these states are of crucial importance in the energy of sunlight). The catabolic (energy-yielding)
metabolism (oxidation-reduction reactions are the pathways described in Chapters 14 through 19 are oxi-
topic of Section 13.4). In many biological oxidations, a dative reaction sequences that result in the transfer of
electrons from fuel molecules, through a series of electron
carriers, to oxygen. The high affinity of O2 for electrons
makes the overall electron-transfer process highly exer-
gonic, providing the energy that drives ATP synthesis—
the central goal of catabolism.
CH2 CH3 Alkane Many of the reactions within these five classes are
CH2 CH2OH Alcohol facilitated by cofactors, in the form of coenzymes and
O
metals (vitamin B12, S-adenosylmethionine, folate, nico-
CH2 C Aldehyde (ketone) tinamide, and iron are some examples). Cofactors bind
H(R) to enzymes—in some cases reversibly, in other cases
almost irreversibly—and confer on them the capacity to
O
promote a particular kind of chemistry (p. 190). Most
CH2 C Carboxylic acid
OH cofactors participate in a narrow range of closely related
reactions. In the following chapters, we will introduce
O C O Carbon dioxide
and discuss each important cofactor at the point where
FIGURE 13–9 The oxidation levels of carbon in biomolecules. Each we first encounter it. The cofactors provide another way
compound is formed by oxidation of the red carbon in the compound to organize the study of biochemical processes, since
shown immediately above. Carbon dioxide is the most highly oxidized the reactions facilitated by a given cofactor generally
form of carbon found in living systems. are mechanistically related.
13.3 Phosphoryl Group Transfers and ATP 517
Biochemical and Chemical Equations Are Not Identical intermediates are common and are stabilized by
adjacent carbonyl groups or, less often, by imines
Biochemists write metabolic equations in a simplified
or certain cofactors.
way, and this is particularly evident for reactions
! A redistribution of electrons can produce
involving ATP. Phosphorylated compounds can exist in
several ionization states and, as we have noted, the dif- internal rearrangements, isomerizations, and
ferent species can bind Mg21. For example, at pH 7 and eliminations. Such reactions include
2 mM Mg21, ATP exists in the forms ATP42 , HATP32 , intramolecular oxidation-reduction, change in
H2ATP22 , MgHATP2, and Mg2ATP. In thinking about cis-trans arrangement at a double bond, and
the biological role of ATP, however, we are not always transposition of double bonds.
interested in all this detail, and so we consider ATP as an ! Homolytic cleavage of covalent bonds to generate
entity made up of a sum of species, and we write its free radicals occurs in some pathways, such as in
hydrolysis as the biochemical equation certain isomerization, decarboxylation, reductase,
and rearrangement reactions.
ATP 1 H2O 88n ADP 1 Pi
! Phosphoryl transfer reactions are an especially
where ATP, ADP, and Pi are sums of species. The cor- important type of group transfer in cells, required
responding standard transformed equilibrium constant, for the activation of molecules for reactions that
K¿eq 5 [ADP4 [Pi 4/[ATP4, depends on the pH and the would otherwise be highly unfavorable.
concentration of free Mg21. Note that H1 and Mg21 do ! Oxidation-reduction reactions involve the loss
not appear in the biochemical equation because they or gain of electrons: one reactant gains
are held constant. Thus a biochemical equation does not electrons and is reduced, while the other loses
necessarily balance H, Mg, or charge, although it does electrons and is oxidized. Oxidation reactions
balance all other elements involved in the reaction generally release energy and are important in
(C, N, O, and P in the equation above). catabolism.
We can write a chemical equation that does balance
for all elements and for charge. For example, when ATP
is hydrolyzed at a pH above 8.5 in the absence of Mg21,
the chemical reaction is represented by
13.3 Phosphoryl Group Transfers and ATP
42 32
ATP 1 H2O 88n ADP 1 HPO22
4 1H 1
Having developed some fundamental principles of
32 energy changes in chemical systems and reviewed the
The corresponding equilibrium constant, K¿eq 5 [ADP ]
common classes of reactions, we can now examine
[HPO22 1
4 ][H ]/[ATP
42
], depends only on temperature,
the energy cycle in cells and the special role of ATP
pressure, and ionic strength.
as the energy currency that links catabolism and
Both ways of writing a metabolic reaction have
anabolism (see Fig. 1–29). Heterotrophic cells obtain
value in biochemistry. Chemical equations are needed
free energy in a chemical form by the catabolism of
when we want to account for all atoms and charges in
nutrient molecules, and they use that energy to make
a reaction, as when we are considering the mechanism
ATP from ADP and Pi. ATP then donates some of its
of a chemical reaction. Biochemical equations are
chemical energy to endergonic processes such as the
used to determine in which direction a reaction will
synthesis of metabolic intermediates and macromol-
proceed spontaneously, given a specified pH and
ecules from smaller precursors, the transport of sub-
[Mg21], or to calculate the equilibrium constant of
stances across membranes against concentration
such a reaction.
gradients, and mechanical motion. This donation of
Throughout this book we use biochemical equa-
energy from ATP generally involves the covalent par-
tions, unless the focus is on chemical mechanism, and
ticipation of ATP in the reaction that is to be driven,
we use values of DG98 and K¿eq as determined at pH 7
with the eventual result that ATP is converted to
and 1 mM Mg21.
ADP and Pi or, in some reactions, to AMP and 2 Pi. We
discuss here the chemical basis for the large free-
energy changes that accompany hydrolysis of ATP
SUMMARY 13.2 Chemical Logic and Common and other high-energy phosphate compounds, and we
show that most cases of energy donation by ATP
Biochemical Reactions
involve group transfer, not simple hydrolysis of ATP.
! Living systems make use of a large number of To illustrate the range of energy transductions in
chemical reactions that can be classified into five which ATP provides the energy, we consider the syn-
general types. thesis of information-rich macromolecules, the trans-
! Carbonyl groups play a special role in reactions port of solutes across membranes, and motion produced
that form or cleave COC bonds. Carbanion by muscle contraction.
O O O FIGURE 13–11 Chemical basis for the large free-energy change associ-
B B B
ated with ATP hydrolysis. 1 The charge separation that results from
"
OO PO O OP OO OP OO O Rib O Adenine
A A A hydrolysis relieves electrostatic repulsion among the four negative
H O O" O" O" ATP4! charges on ATP. 2 The product inorganic phosphate (Pi) is stabilized by
H formation of a resonance hybrid, in which each of the four phosphorus–
oxygen bonds has the same degree of double-bond character and the
O 1 hydrogen ion is not permanently associated with any one of the oxygens.
hydrolysis,
B (Some degree of resonance stabilization also occurs in phosphates
with relief
"
O OP OOH of charge involved in ester or anhydride linkages, but fewer resonance forms are
A repulsion
Pi O" possible than for Pi.) A third factor (not shown) that favors ATP hydroly-
sis is the greater degree of solvation (hydration) of the products Pi and
resonance
2 stabilization
ADP relative to ATP, which further stabilizes the products relative to the
reactants.
'" 3"
O
A
' "OOP OO ' " H!
A
O
'"
O O
B B
HO OP OO OP OO O Rib O Adenine terminal phosphoric acid anhydride (phosphoanhy-
A A dride) bond in ATP separates one of the three nega-
O" O" ADP2!
tively charged phosphates and thus relieves some of
the electrostatic repulsion in ATP; the Pi released is
ionization stabilized by the formation of several resonance forms
not possible in ATP.
O O The free-energy change for ATP hydrolysis is
B B
H ! O OP OO OP O OO Rib O Adenine
! " 230.5 kJ/mol under standard conditions, but the actual
A A free energy of hydrolysis (DG) of ATP in living cells is
O" O" ADP3! very different: the cellular concentrations of ATP,
ADP, and Pi are not identical and are much lower
ATP4" ! H2O ADP 3" ! HPO2"
4 ! H
!
than the 1.0 M of standard conditions (Table 13–5).
$G%& # "30.5 kJ/mol Furthermore, Mg21 in the cytosol binds to ATP and
ADP (Fig. 13–12), and for most enzymatic reactions
that involve ATP as phosphoryl group donor, the true
substrate is MgATP22. The relevant DG98 is therefore
The Free-Energy Change for ATP Hydrolysis that for MgATP22 hydrolysis. We can calculate DG for
Is Large and Negative ATP hydrolysis using data such as those in Table 13–5.
Figure 13–11 summarizes the chemical basis for The actual free energy of hydrolysis of ATP under
the relatively large, negative, standard free energy of intracellular conditions is often called its phosphoryl-
hydrolysis of ATP. The hydrolytic cleavage of the ation potential, DGp.
TABLE 13–5 Adenine Nucleotide, Inorganic Phosphate, and

Phosphocreatine Concentrations in Some Cells O O O
B B B
Concentration (mM)* "
O OP OO OP O OO PO OO Rib O Adenine
A A A
†
ATP ADP AMP Pi PCr O" O"
ø ø2!
O" MgATP2!
Mg
Rat hepatocyte 3.38 1.32 0.29 4.8 0
Rat myocyte 8.05 0.93 0.04 8.05 28 O O
B B
Rat neuron 2.59 0.73 0.06 2.72 4.7 "
OO PO OOPO OO Rib O Adenine
A A
Human erythrocyte 2.25 0.25 0.02 1.65 0 O" O" MgADP!
ø ø2!
E. coli cell 7.90 1.04 0.82 7.9 0 Mg
*For erythrocytes the concentrations are those of the cytosol (human erythrocytes lack a nucleus and FIGURE 13–12 Mg21 and ATP. Formation of Mg21
mitochondria). In the other types of cells the data are for the entire cell contents, although the cytosol and the complexes partially shields the negative charges and
mitochondria have very different concentrations of ADP. PCr is phosphocreatine, discussed on p. 526. influences the conformation of the phosphate groups
†
This value reflects total concentration; the true value for free ADP may be much lower (p. 519). in nucleotides such as ATP and ADP.
WORKED EXAMPLE 13–2 Calculation of DGp

Calculate the actual free energy of hydrolysis of ATP, DGp, in human erythrocytes.
The standard free energy of hydrolysis of ATP is 230.5 kJ/mol, and the concentra-
tions of ATP, ADP, and Pi in erythrocytes are as shown in Table 13–5. Assume that
the pH is 7.0 and the temperature is 37 8C (body temperature). What does this
reveal about the amount of energy required to synthesize ATP under the same cel-
lular conditions?
Solution: The concentrations of ATP, ADP, and Pi in human erythrocytes are 2.25,
0.25, and 1.65 mM, respectively. The actual free energy of hydrolysis of ATP under
these conditions is given by the relationship (see Eqn 13–4)
[ADP][Pi]
¢Gp 5 ¢G¿8 1 RT ln
[ATP]
Substituting the appropriate values we get
(0.25 3 1023)(1.65 3 1023)
¢Gp 5 230.5 kJ/mol 1 c (8.315 J/mol ? K)(310 K) ln d
(2.25 3 1023)
24
5 230.5 kJ/mol 1 (2.58 kJ/mol) ln 1.8 3 10
5 230.5 kJ/mol 1 (2.58 kJ/mol)(28.6)
5 230.5 kJ/mol 2 22 kJ/mol
5 252 kJ/mol
(Note that the final answer has been rounded to the correct number of significant
figures (52.5 rounded to 52), following rules for rounding a number that ends in a
5 to the nearest even number.) Thus DGp, the actual free-energy change for ATP
hydrolysis in the intact erythrocyte (252 kJ/mol), is much larger than the standard
free-energy change (230.5 kJ/mol). By the same token, the free energy required to
synthesize ATP from ADP and Pi under the conditions prevailing in the erythrocyte
would be 52 kJ/mol.
Because the concentrations of ATP, ADP, and Pi In the following discussions we use the DG98 value
differ from one cell type to another, DGp for ATP like- for ATP hydrolysis because this allows comparison, on
wise differs among cells. Moreover, in any given cell, the same basis, with the energetics of other cellular reac-
DGp can vary from time to time, depending on the meta- tions. Always keep in mind, however, that in living cells
bolic conditions and how they influence the concentra- DG is the relevant quantity—for ATP hydrolysis and all
tions of ATP, ADP, Pi, and H1 (pH). We can calculate other reactions—and may be quite different from DG98.
the actual free-energy change for any given metabolic Here we must make an important point about cellu-
reaction as it occurs in a cell, providing we know the lar ATP levels. We have shown (and will discuss further)
concentrations of all the reactants and products and how the chemical properties of ATP make it a suitable
other factors (such as pH, temperature, and [Mg21]) form of energy currency in cells. But it is not merely the
that may affect the actual free-energy change. molecule’s intrinsic chemical properties that give it this
To further complicate the issue, the total concen- ability to drive metabolic reactions and other energy-
trations of ATP, ADP, Pi, and H1 in a cell may be sub- requiring processes. Even more important is that, in the
stantially higher than the free concentrations, which course of evolution, there has been a very strong selec-
are the thermodynamically relevant values. The differ- tive pressure for regulatory mechanisms that hold cel-
ence is due to tight binding of ATP, ADP, and Pi to lular ATP concentrations far above the equilibrium
cellular proteins. For example, the free [ADP] in resting concentrations for the hydrolysis reaction. When the
muscle has been variously estimated at between 1 and ATP level drops, not only does the amount of fuel
37 #M. Using the value 25 #M in Worked Example 13–2, decrease, but the fuel itself loses its potency: DG for its
we would get a DGp of 264 kJ/mol. Calculation of the hydrolysis (that is, its phosphorylation potential, DGp) is
exact value of DGp, however, is perhaps less instruc- diminished. As our discussions of the metabolic pathways
tive than the generalization we can make about actual that produce and consume ATP will show, living cells
free-energy changes: in vivo, the energy released by have developed elaborate mechanisms—often at what
ATP hydrolysis is greater than the standard free-ener- might seem to us the expense of efficiency and common
gy change, DG98. sense—to maintain high concentrations of ATP.
FIGURE 13–13 Hydrolysis of phosphoenolpyru- O "

O
G J
vate (PEP). Catalyzed by pyruvate kinase, this O P H2O O O
reaction is followed by spontaneous tautomeriza-
J D G " J J
"
O OC O O "
O OC OH O OC
"
O
tion of the product, pyruvate. Tautomerization is G D hydrolysis G D G J
C C tautomerization C
not possible in PEP, and thus the products of B Pi B A
hydrolysis are stabilized relative to the reactants. CH2 CH2 CH3
Resonance stabilization of Pi also occurs, as PEP Pyruvate Pyruvate
shown in Figure 13–11. (enol form) (keto form)
PEP3" ! H2O pyruvate" ! HPO2"

4
$G%& # "61.9 kJ/mol
Other Phosphorylated Compounds and Thioesters Also can, again, be explained in terms of the structure of
reactant and products. When H2O is added across the
Have Large Free Energies of Hydrolysis
anhydride bond of 1,3-bisphosphoglycerate, one of the
Phosphoenolpyruvate (PEP; Fig. 13–13) contains a direct products, 3-phosphoglyceric acid, can lose a proton
phosphate ester bond that undergoes hydrolysis to to give the carboxylate ion, 3-phosphoglycerate, which
yield the enol form of pyruvate, and this direct prod- has two equally probable resonance forms (Fig. 13–14).
uct can tautomerize to the more stable keto form. Removal of the direct product (3-phosphoglyceric acid)
Because the reactant (PEP) has only one form (enol) and formation of the resonance-stabilized ion favor the
and the product (pyruvate) has two possible forms, forward reaction.
the product is stabilized relative to the reactant. This In phosphocreatine (Fig. 13–15), the PON bond
is the greatest contributing factor to the high standard can be hydrolyzed to generate free creatine and Pi. The
free energy of hydrolysis of phosphoenolpyruvate: release of Pi and the resonance stabilization of creatine
DG98 5 261.9 kJ/mol. favor the forward reaction. The standard free-energy
Another three-carbon compound, 1,3-bisphospho- change of phosphocreatine hydrolysis is again large,
glycerate (Fig. 13–14), contains an anhydride bond 243.0 kJ/mol.
between the C-1 carboxyl group and a number that In all these phosphate-releasing reactions, the sev-
ends in a phosphoric acid. Hydrolysis of this acyl eral resonance forms available to Pi (Fig. 13–11) stabi-
phosphate is accompanied by a large, negative, standard lize this product relative to the reactant, contributing to
free-energy change (DG98 5 249.3 kJ/mol), which an already negative free-energy change. Table 13–6 lists
"
O O
G J
P
D G " '" '"
O O O O OH O O resonance
M D M D G D stabilization
1C C C
A Pi A H! A
2 CHOH CHOH CHOH
A A A
FIGURE 13–14 Hydrolysis of 1,3-bisphos- 3 CH2 CH2 CH2
phoglycerate. The direct product of hydro- A H2O A A
lysis is 3-phosphoglyceric acid, with an O O O
A hydrolysis A ionization A
undissociated carboxylic acid. Its dissocia- "
OO PP O "
O O PP O "
OO PP O
tion allows resonance structures that stabi- A A A
O" O" O"
lize the product relative to the reactants.
Resonance stabilization of Pi further con- 1,3-Bisphosphoglycerate 3-Phosphoglyceric acid 3-Phosphoglycerate
tributes to the negative free-energy change.
1,3-Bisphosphoglycerate4" ! H2O 3-phosphoglycerate3" ! HPO2"
4 !H
!
$G%& # "49.3 kJ/mol
COO" COO" COO"

A A A
'!
O CH2 H2O CH2 CH2
B H A A H2N A
FIGURE 13–15 Hydrolysis of phosphocre-
"
O OP ONOCONOCH3 H2NOCONOCH3 C NOCH3
A B hydrolysis B resonance '!
atine. Breakage of the PON bond in phos- stabilization H2N
O " !
NH2 Pi
!
NH2 '!
phocreatine produces creatine, which is Phosphocreatine Creatine
stabilized by formation of a resonance
2"
hybrid. The other product, Pi, is also reso- Phosphocreatine ! H2O creatine ! HPO2"
4
nance stabilized. $G%& # "43.0 kJ/mol

O
TABLE 13–6 Standard Free Energies of Hydrolysis of CH3 OC
J
Acetyl-CoA
Some Phosphorylated Compounds and G
S-CoA
Acetyl-CoA (a Thioester)
H2O hydrolysis
DG98 CoASH
(kJ/mol) (kcal/mol)
O
J
Phosphoenolpyruvate 261.9 214.8 CH3 OC Acetic acid
G
1,3-Bisphosphoglycerate OH
(n 3-phosphoglycerate 1 Pi) 249.3 211.8
ionization
Phosphocreatine 243.0 210.3
H!
ADP (n AMP 1 Pi) 232.8 27.8
O'"
ATP (n ADP 1 Pi) 230.5 27.3 D
CH3 OC Acetate
ATP (n AMP 1 PPi) 245.6 210.9 G '"
O
AMP (n adenosine 1 Pi) 214.2 23.4 resonance
stabilization
PPi (n 2Pi) 219.2 24.0
Glucose 3-phosphate 220.9 25.0 Acetyl-CoA ! H2O acetate" ! CoA ! H!
$G%& # "31.4 kJ/mol
Fructose 6-phosphate 215.9 23.8
Glucose 6-phosphate 213.8 23.3 FIGURE 13–16 Hydrolysis of acetyl-coenzyme A. Acetyl-CoA is a
Glycerol 3-phosphate 29.2 22.2 thioester with a large, negative, standard free energy of hydrolysis.
Thioesters contain a sulfur atom in the position occupied by an oxygen
Acetyl-CoA 231.4 27.5
atom in oxygen esters. The complete structure of coenzyme A (CoA, or
Source: Data mostly from Jencks, W.P. (1976) in Handbook of Biochemistry and Molecular CoASH) is shown in Figure 8–38.
Biology, 3rd edn (Fasman, G.D., ed.), Physical and Chemical Data, Vol. 1, pp. 296–304, CRC
Press, Boca Raton, FL. The value for the free energy of hydrolysis of PPi is from Frey, P.A. &
Arabshahi, A. (1995) Standard free-energy change for the hydrolysis of the
$-%-phosphoanhydride bridge in ATP. Biochemistry 34, 11,307-11,310. reactant and its hydrolysis products, which are
resonance-stabilized, is greater for thioesters than for
comparable oxygen esters (Fig. 13–17). In both cases,
the standard free energies of hydrolysis for some bio- hydrolysis of the ester generates a carboxylic acid,
logically important phosphorylated compounds. which can ionize and assume several resonance forms.
Thioesters, in which a sulfur atom replaces the Together, these factors result in the large, negative DG98
usual oxygen in the ester bond, also have large, negative, (231.4 kJ/mol) for acetyl-CoA hydrolysis.
standard free energies of hydrolysis. Acetyl-coenzyme A, To summarize, for hydrolysis reactions with large,
or acetyl-CoA (Fig. 13–16), is one of many thioesters negative, standard free-energy changes, the products
important in metabolism. The acyl group in these com- are more stable than the reactants for one or more of the
pounds is activated for transacylation, condensation, or following reasons: (1) the bond strain in reactants due to
oxidation-reduction reactions. Thioesters undergo much electrostatic repulsion is relieved by charge separation,
less resonance stabilization than do oxygen esters; as for ATP; (2) the products are stabilized by ionization,
consequently, the difference in free energy between the as for ATP, acyl phosphates, and thioesters; (3) the
Thioester Extra stabilization of

oxygen ester by resonance
O
J
CH3 OC
G Oxygen
S OR
ester Resonance '"
O stabilization O
Free energy, G
J FIGURE 13–17 Free energy of hydrolysis for

CH3 OC CH3 C
$G for G thioesters and oxygen esters. The products of
thioester O OR OO R
hydrolysis '! both types of hydrolysis reaction have about the
$G for oxygen same free-energy content (G), but the thioester
ester hydrolysis has a higher free-energy content than the oxygen
ester. Orbital overlap between the O and C
O O
J J atoms allows resonance stabilization in oxygen
CH3 OC ! ROSH CH3 OC ! RO OH
G G esters; orbital overlap between S and C atoms is
OH OH poorer and provides little resonance stabilization.
products are stabilized by isomerization (tautomeriza- acid residue in an enzyme, becoming covalently attached
tion), as for PEP; and/or (4) the products are stabilized to the substrate or the enzyme and raising its free-
by resonance, as for creatine released from phosphocre- energy content. Then, in a second step, the phosphate-
atine, carboxylate ion released from acyl phosphates and containing moiety transferred in the first step is
thioesters, and phosphate (Pi) released from anhydride displaced, generating Pi, PPi, or AMP. Thus ATP partici-
or ester linkages. pates covalently in the enzyme-catalyzed reaction to
which it contributes free energy.
Some processes do involve direct hydrolysis of
ATP Provides Energy by Group Transfers, ATP (or GTP), however. For example, noncovalent
Not by Simple Hydrolysis binding of ATP (or GTP), followed by its hydrolysis to
Throughout this book you will encounter reactions or ADP (or GDP) and Pi, can provide the energy to cycle
processes for which ATP supplies energy, and the con- some proteins between two conformations, producing
tribution of ATP to these reactions is commonly indi- mechanical motion. This occurs in muscle contraction
cated as in Figure 13–18a, with a single arrow showing (see Fig. 5–31), and in the movement of enzymes along
the conversion of ATP to ADP and Pi (or, in some cases, DNA (see Fig. 25–31) or of ribosomes along messenger
of ATP to AMP and pyrophosphate, PPi). When written RNA (see Fig. 27–31). The energy-dependent reactions
this way, these reactions of ATP seem to be simple catalyzed by helicases, RecA protein, and some topo-
hydrolysis reactions in which water displaces Pi (or isomerases (Chapter 25) also involve direct hydrolysis
PPi), and one is tempted to say that an ATP-dependent of phosphoanhydride bonds. The AAA1 ATPases
reaction is “driven by the hydrolysis of ATP.” This is not involved in DNA replication and other processes
the case. ATP hydrolysis per se usually accomplishes described in Chapter 25 use ATP hydrolysis to cycle
nothing but the liberation of heat, which cannot drive a associated proteins between active and inactive forms.
chemical process in an isothermal system. A single reac- GTP-binding proteins that act in signaling pathways
tion arrow such as that in Figure 13–18a almost invari- directly hydrolyze GTP to drive conformational changes
ably represents a two-step process (Fig. 13–18b) in that terminate signals triggered by hormones or by
which part of the ATP molecule, a phosphoryl or pyro- other extracellular factors (Chapter 12).
phosphoryl group or the adenylate moiety (AMP), is The phosphate compounds found in living organ-
first transferred to a substrate molecule or to an amino isms can be divided somewhat arbitrarily into two
groups, based on their standard free energies of hy-
drolysis (Fig. 13–19). “High-energy” compounds have
(a) Written as a one-step reaction a DG98 of hydrolysis more negative than 225 kJ/mol;
COO! COO! “low-energy” compounds have a less negative DG98.
"A "A Based on this criterion, ATP, with a DG98 of hydrolysis
H3NOCH ATP ADP " Pi H3NOCH
A A of 230.5 kJ/mol (27.3 kcal/mol), is a high-energy com-
CH2 CH2 pound; glucose 6-phosphate, with a DG98 of hydrolysis
A " NH3 A
CH2 CH2 of 213.8 kJ/mol (23.3 kcal/mol), is a low-energy
A A
C C compound.
J G ! J G The term “high-energy phosphate bond,” long used
O O O NH2
Glutamate Glutamine by biochemists to describe the POO bond broken in
hydrolysis reactions, is incorrect and misleading as it
(b) Actual two-step reaction
wrongly suggests that the bond itself contains the energy.
NH3 In fact, the breaking of all chemical bonds requires an
COO! input of energy. The free energy released by hydrolysis
"A
ATP H3NOCH of phosphate compounds does not come from the spe-
1 A 2
CH2 cific bond that is broken; it results from the products of
ADP A Pi
the reaction having a lower free-energy content than the
CH2
A reactants. For simplicity, we will sometimes use the term
C “high-energy phosphate compound” when referring to
J G
O O O ATP or other phosphate compounds with a large, nega-
G J
P
Enzyme-bound D G ! tive, standard free energy of hydrolysis.
glutamyl phosphate O O
!
As is evident from the additivity of free-energy
changes of sequential reactions (see Section 13.1), any
FIGURE 13–18 ATP hydrolysis in two steps. (a) The contribution of ATP phosphorylated compound can be synthesized by cou-
to a reaction is often shown as a single step, but is almost always a two- pling the synthesis to the breakdown of another phos-
step process. (b) Shown here is the reaction catalyzed by ATP-depen- phorylated compound with a more negative free energy
dent glutamine synthetase. 1 A phosphoryl group is transferred from of hydrolysis. For example, because cleavage of Pi from
ATP to glutamate, then 2 the phosphoryl group is displaced by NH3 phosphoenolpyruvate releases more energy than is
and released as Pi. needed to drive the condensation of Pi with ADP, the
Phosphoenolpyruvate
FIGURE 13–19 Ranking of biological phosphate compounds by standard
!70
COO! free energies of hydrolysis. This shows the flow of phosphoryl groups,
1,3-Bisphosphoglycerate A represented by P , from high-energy phosphoryl group donors via ATP to
CO OO P
3
CH2OOO P B acceptor molecules (such as glucose and glycerol) to form their low-energy
!60 A CH2
2 phosphate derivatives. (The location of each compound’s donor phosphoryl
CHOH
A Phosphocreatine group along the scale approximately indicates the DG98 of hydrolysis.)
1
MCD COO! This flow of phosphoryl groups, catalyzed by kinases, proceeds with an
!50 A
O OO P overall loss of free energy under intracellular conditions. Hydrolysis of
CH2
H A low-energy phosphate compounds releases Pi, which has an even lower
P ONOCONOCH3 phosphoryl group transfer potential (as defined in the text).
!40 B
"
NH2
ATP
Adenine O Rib O P O P O P
!30 High-energy
compounds
Low-energy
!20 compounds
Glucose 6- P
!10
Glycerol- P
0 Pi
direct donation of a phosphoryl group from PEP to ADP high-energy phosphate compounds produced by
is thermodynamically feasible: catabolism to compounds such as glucose, converting
DG98 (kJ/mol) them into more reactive species. ATP thus serves as the
universal energy currency in all living cells.
(1) PEP 1 H2O 88n pyruvate 1 Pi 261.9
One more chemical feature of ATP is crucial to its
(2) ADP 1 Pi 88n ATP 1 H2O 130.5
role in metabolism: although in aqueous solution ATP is
Sum: PEP 1 ADP 88n pyruvate 1 ATP 231.4 thermodynamically unstable and is therefore a good
phosphoryl group donor, it is kinetically stable. Because
Notice that while the overall reaction is represented as of the huge activation energies (200 to 400 kJ/mol)
the algebraic sum of the first two reactions, the overall required for uncatalyzed cleavage of its phosphoanhy-
reaction is actually a third, distinct reaction that does not dride bonds, ATP does not spontaneously donate phos-
involve Pi; PEP donates a phosphoryl group directly to phoryl groups to water or to the hundreds of other poten-
ADP. We can describe phosphorylated compounds as tial acceptors in the cell. Only when specific enzymes are
having a high or low phosphoryl group transfer potential, present to lower the energy of activation does phosphoryl
on the basis of their standard free energies of hydrolysis group transfer from ATP proceed. The cell is therefore
(as listed in Table 13–6). The phosphoryl group transfer able to regulate the disposition of the energy carried by
potential of PEP is very high, that of ATP is high, and that ATP by regulating the various enzymes that act on it.
of glucose 6-phosphate is low (Fig. 13–19).
Much of catabolism is directed toward the synthesis
of high-energy phosphate compounds, but their forma-
ATP Donates Phosphoryl, Pyrophosphoryl,
tion is not an end in itself; they are the means of activat- and Adenylyl Groups
ing a very wide variety of compounds for further The reactions of ATP are generally SN2 nucleophilic
chemical transformation. The transfer of a phosphoryl displacements (see Section 13.2) in which the nucleo-
group to a compound effectively puts free energy into phile may be, for example, the oxygen of an alcohol or
that compound, so that it has more free energy to give carboxylate, or a nitrogen of creatine or of the side
up during subsequent metabolic transformations. We chain of arginine or histidine. Each of the three phos-
described above how the synthesis of glucose 6-phosphate phates of ATP is susceptible to nucleophilic attack
is accomplished by phosphoryl group transfer from (Fig. 13–20), and each position of attack yields a
ATP. In the next chapter we see how this phosphoryla- different type of product.
tion of glucose activates, or “primes,” the glucose for Nucleophilic attack by an alcohol on the & phos-
catabolic reactions that occur in nearly every living cell. phate (Fig. 13–20a) displaces ADP and produces a new
Because of its intermediate position on the scale of phosphate ester. Studies with 18O-labeled reactants have
group transfer potential, ATP can carry energy from shown that the bridge oxygen in the new compound is
FIGURE 13–20 Nucleophilic displacement reactions of Three positions on ATP for attack by the nucleophile R18O
:
ATP. Any of the three P atoms (#, $, or %) may serve as " # $
the electrophilic target for nucleophilic attack—in this case, O O O
by the labeled nucleophile R218O:. The nucleophile may be !O P O P O P O Rib Adenine
an alcohol (ROH), a carboxyl group (RCOO2), or a phos-
phoanhydride (a nucleoside mono- or diphosphate, for O! O! O!
:
example). (a) When the oxygen of the nucleophile attacks R18O R18O R18O
the % position, the bridge oxygen of the product is labeled,
indicating that the group transferred from ATP is a phos-
phoryl (2PO22 22
3 ), not a phosphate (2OPO3 ). (b) Attack
O O O O
on the $ position displaces AMP and leads to the transfer of
a pyrophosphoryl (not pyrophosphate) group to the nucleo- R18O P O! R18O P O P O! R18O P O Rib Adenine
phile. (c) Attack on the a position displaces PPi and trans- O! O! O! O!
fers the adenylyl group to the nucleophile. % % %
ADP AMP PPi
Phosphoryl Pyrophosphoryl Adenylyl
transfer transfer transfer
(a) (b) (c)
derived from the alcohol, not from ATP; the group trans- (fatty acyl adenylate) and liberating PPi. The thiol
ferred from ATP is therefore a phosphoryl (2PO22 3 ), not group of coenzyme A then displaces the adenylyl group
a phosphate (2OPO22 3 ). Phosphoryl group transfer from and forms a thioester with the fatty acid. The sum of
ATP to glutamate (Fig. 13–18) or to glucose (p. 219) these two reactions is energetically equivalent to the
involves attack at the % position of the ATP molecule. exergonic hydrolysis of ATP to AMP and PPi
Attack at the $ phosphate of ATP displaces AMP and (¢G¿8 5 245.6 kJ/mol) and the endergonic formation
transfers a pyrophosphoryl (not pyrophosphate) group to of fatty acyl–CoA (¢G¿8 5 31.4 kJ/mol). The formation
the attacking nucleophile (Fig. 13–20b). For example, the of fatty acyl–CoA is made energetically favorable by
formation of 5-phosphoribosyl-1-pyrophosphate (p. 892), hydrolysis of the PPi by inorganic pyrophosphatase.
a key intermediate in nucleotide synthesis, results from Thus, in the activation of a fatty acid, both phosphoan-
attack of an OOH of the ribose on the $ phosphate. hydride bonds of ATP are broken. The resulting ¢G¿8 is
Nucleophilic attack at the # position of ATP displaces the sum of the ¢G¿8 values for the breakage of these
PPi and transfers adenylate (59-AMP) as an adenylyl bonds, or 245.6 kJ/mol 1 (219.2) kJ/mol:
group (Fig. 13–20c); the reaction is an adenylylation
ATP 1 2H2O 88n AMP 1 2Pi ¢G¿8 5 264.8 kJ/mol
(a-den9-i-li-la9-shun, one of the most ungainly words in
the biochemical language). Notice that hydrolysis of the The activation of amino acids before their polymer-
#–$ phosphoanhydride bond releases considerably more ization into proteins (see Fig. 27–19) is accomplished
energy (,46 kJ/mol) than hydrolysis of the $–% bond by an analogous set of reactions in which a transfer RNA
(,31 kJ/mol) (Table 13–6). Furthermore, the PPi formed molecule takes the place of coenzyme A. An interesting
as a byproduct of the adenylylation is hydrolyzed to two use of the cleavage of ATP to AMP and PPi occurs in the
Pi by the ubiquitous enzyme inorganic pyrophospha- firefly, which uses ATP as an energy source to produce
tase, releasing 19 kJ/mol and thereby providing a further light flashes (Box 13–1).
energy “push” for the adenylylation reaction. In effect,
both phosphoanhydride bonds of ATP are split in the
overall reaction. Adenylylation reactions are therefore
Assembly of Informational Macromolecules
thermodynamically very favorable. When the energy of Requires Energy
ATP is used to drive a particularly unfavorable metabolic When simple precursors are assembled into high molec-
reaction, adenylylation is often the mechanism of energy ular weight polymers with defined sequences (DNA,
coupling. Fatty acid activation is a good example of this RNA, proteins), as described in detail in Part III, energy
energy-coupling strategy. is required both for the condensation of monomeric
The first step in the activation of a fatty acid— units and for the creation of ordered sequences. The
either for energy-yielding oxidation or for use in the precursors for DNA and RNA synthesis are nucleoside
synthesis of more complex lipids—is the formation of its triphosphates, and polymerization is accompanied by
thiol ester (see Fig. 17–5). The direct condensation of a cleavage of the phosphoanhydride linkage between the
fatty acid with coenzyme A is endergonic, but the for- # and $ phosphates, with the release of PPi (Fig.
mation of fatty acyl–CoA is made exergonic by stepwise 13–20). The moieties transferred to the growing poly-
removal of two phosphoryl groups from ATP. First, mer in these reactions are adenylate (AMP), guanylate
adenylate (AMP) is transferred from ATP to the car- (GMP), cytidylate (CMP), or uridylate (UMP) for RNA
boxyl group of the fatty acid, forming a mixed anhydride synthesis, and their deoxy analogs (with TMP in place
BOX 13–1 Firefly Flashes: Glowing Reports of ATP

Bioluminescence requires considerable amounts of process is accompanied by emission of light. The color
energy. In the firefly, ATP is used in a set of reactions of the light flash differs with the firefly species and
that converts chemical energy into light energy. In the seems to be determined by differences in the struc-
1950s, from many thousands of fireflies collected by ture of the luciferase. Luciferin is regenerated from
children in and around Baltimore, William McElroy oxyluciferin in a subsequent series of reactions.
and his colleagues at the Johns Hopkins University In the laboratory, pure firefly luciferin and lucif-
isolated the principal biochemical components: luciferase are used to measure minute quantities of ATP
erin, a complex carboxylic acid, and luciferase, an by the intensity of the light flash produced. As little
enzyme. The generation of a light flash requires acti- as a few picomoles (10212 mol) of ATP can be mea-
vation of luciferin by an enzymatic reaction involving sured in this way. Next-gen pyrosequencing of DNA
pyrophosphate cleavage of ATP to form luciferyl relies on flashes of light from the luciferin-luciferase
adenylate (Fig. 1). In the presence of molecular oxy- reaction to detect the presence of ATP after addi-
gen and luciferase, the luciferin undergoes a multi- tion of nucleotides to a growing strand of DNA (see
step oxidative decarboxylation to oxyluciferin. This Fig. 9–25).
H O"
N N A
C O P O Rib Adenine
H
HO S S O O
H AMP
Luciferyl adenylate
PPi
O2
luciferase
ATP light
H
N N
COO"
H CO2 ! AMP
HO S S
H
Luciferin N N O
The firefly, a beetle of the Lampyridae family. S S

HO
regenerating Oxyluciferin
reactions
FIGURE 1 Important components in the firefly bioluminescence cycle.
of UMP) for DNA synthesis. As noted above, the activa- the Na1K1 ATPase. The transport of Na1 and K1 is
tion of amino acids for protein synthesis involves the driven by cyclic phosphorylation and dephosphorylation
donation of adenylyl groups from ATP, and we shall see of the transporter protein, with ATP as the phosphoryl
in Chapter 27 that several steps of protein synthesis on group donor. Na1-dependent phosphorylation of the
the ribosome are also accompanied by GTP hydrolysis. Na1K1 ATPase forces a change in the protein's confor-
In all these cases, the exergonic breakdown of a nucleo- mation, and K1-dependent dephosphorylation favors
side triphosphate is coupled to the endergonic process return to the original conformation. Each cycle in the
of synthesizing a polymer of a specific sequence. transport process results in the conversion of ATP to
ADP and Pi, and it is the free-energy change of ATP
hydrolysis that drives the cyclic changes in protein con-
ATP Energizes Active Transport and Muscle Contraction formation that result in the electrogenic pumping of
ATP can supply the energy for transporting an ion or a Na1 and K1. Note that in this case ATP interacts cova-
molecule across a membrane into another aqueous com- lently by phosphoryl group transfer to the enzyme, not
partment where its concentration is higher (see Fig. the substrate.
11–38). Transport processes are major consumers of In the contractile system of skeletal muscle cells,
energy; in human kidney and brain, for example, as myosin and actin are specialized to transduce the chem-
much as two-thirds of the energy consumed at rest is ical energy of ATP into motion (see Fig. 5–31). ATP
used to pump Na1 and K1 across plasma membranes via binds tightly but noncovalently to one conformation of
myosin, holding the protein in that conformation. When tion to the right, with the net formation of NTPs and
myosin catalyzes the hydrolysis of its bound ATP, the dNTPs. The enzyme actually catalyzes a two-step phos-
ADP and Pi dissociate from the protein, allowing it to phoryl group transfer, which is a classic case of a double-
relax into a second conformation until another mole- displacement (Ping-Pong) mechanism (Fig. 13–21;
cule of ATP binds. The binding and subsequent hydro- see also Fig. 6–13b). First, phosphoryl group transfer
lysis of ATP (by myosin ATPase) provide the energy from ATP to an active-site His residue produces a phos-
that forces cyclic changes in the conformation of the phoenzyme intermediate; then the phosphoryl group is
myosin head. The change in conformation of many indi- transferred from the P –His residue to an NDP accep-
vidual myosin molecules results in the sliding of myosin tor. Because the enzyme is nonspecific for the base in
fibrils along actin filaments (see Fig. 5–30), which the NDP and works equally well on dNDPs and NDPs,
translates into macroscopic contraction of the muscle it can synthesize all NTPs and dNTPs, given the corre-
fiber. As we noted earlier, this production of mechani- sponding NDPs and a supply of ATP.
cal motion at the expense of ATP is one of the few Phosphoryl group transfers from ATP result in an
cases in which ATP hydrolysis per se, rather than accumulation of ADP; for example, when muscle is con-
group transfer from ATP, is the source of the chemical tracting vigorously, ADP accumulates and interferes
energy in a coupled process. with ATP-dependent contraction. During periods of
intense demand for ATP, the cell lowers the ADP con-
Transphosphorylations between Nucleotides centration, and at the same time replenishes ATP, by
the action of adenylate kinase:
Occur in All Cell Types
Mg21
Although we have focused on ATP as the cell's energy 2ADP 3::4 ATP 1 AMP ¢G¿8 < 0
currency and donor of phosphoryl groups, all other
nucleoside triphosphates (GTP, UTP, and CTP) and all This reaction is fully reversible, so after the intense
deoxynucleoside triphosphates (dATP, dGTP, dTTP, demand for ATP ends, the enzyme can recycle AMP by
and dCTP) are energetically equivalent to ATP. The converting it to ADP, which can then be phosphorylated
standard free-energy changes associated with hydroly- to ATP in mitochondria. A similar enzyme, guanylate
sis of their phosphoanhydride linkages are very nearly kinase, converts GMP to GDP at the expense of ATP. By
identical with those shown in Table 13–6 for ATP. In pathways such as these, energy conserved in the cata-
preparation for their various biological roles, these bolic production of ATP is used to supply the cell with
other nucleotides are generated and maintained as the all required NTPs and dNTPs.
nucleoside triphosphate (NTP) forms by phosphoryl Phosphocreatine (PCr; Fig. 13–15), also called cre-
group transfer to the corresponding nucleoside diphos- atine phosphate, serves as a ready source of phosphoryl
phates (NDPs) and monophosphates (NMPs). groups for the quick synthesis of ATP from ADP. The
ATP is the primary high-energy phosphate com- PCr concentration in skeletal muscle is approximately
pound produced by catabolism, in the processes of 30 mM, nearly 10 times the concentration of ATP, and in
glycolysis, oxidative phosphorylation, and, in photosyn- other tissues such as smooth muscle, brain, and kidney
thetic cells, photophosphorylation. Several enzymes [PCr] is 5 to 10 mM. The enzyme creatine kinase cata-
then carry phosphoryl groups from ATP to the other lyzes the reversible reaction
nucleotides. Nucleoside diphosphate kinase, found Mg21
in all cells, catalyzes the reaction ADP 1 PCr 3::4 ATP 1 Cr ¢G¿8 5 212.5 kJ/mol
21
Mg
ATP 1 NDP (or dNDP) 3::4 ADP 1 NTP (or dNTP) When a sudden demand for energy depletes ATP, the
¢G¿8 < 0 PCr reservoir is used to replenish ATP at a rate consid-
erably faster than ATP can be synthesized by catabolic
Although this reaction is fully reversible, the relatively pathways. When the demand for energy slackens, ATP
high [ATP]/[ADP] ratio in cells normally drives the reac- produced by catabolism is used to replenish the PCr
Adenosine P P P Enz His Nucleoside P P P

(ATP) (any NTP or dNTP)
Ping Pong
Adenosine P P Enz His P Nucleoside P P

(ADP) (any NDP or dNDP)
FIGURE 13–21 Ping-Pong mechanism of nucleoside diphosphate kinase. The enzyme binds its
first substrate (ATP in our example), and a phosphoryl group is transferred to the side chain of a
His residue. ADP departs, and another nucleoside (or deoxynucleoside) diphosphate replaces it,
and this is converted to the corresponding triphosphate by transfer of the phosphoryl group from
the phosphohistidine residue.
reservoir by reversal of the creatine kinase reaction (see in a wide variety of bacteria, including many pathogenic
Box 23–2). Organisms in the lower phyla employ other species.
PCr-like molecules (collectively called phosphagens) In bacteria, elevated levels of polyP have been
as phosphoryl reservoirs. shown to promote expression of genes involved in adap-
tation of the organism to conditions of starvation or
other threats to survival. In Escherichia coli, for exam-
Inorganic Polyphosphate Is a Potential ple, polyP accumulates when cells are starved for amino
Phosphoryl Group Donor acids or Pi, and this accumulation confers a survival
Inorganic polyphosphate, polyP (or (polyP)n, where n advantage. Deletion of the genes for polyphosphate
is the number of orthophosphate residues), is a linear kinases diminishes the ability of certain pathogenic bac-
polymer composed of many tens or hundreds of Pi resi- teria to invade animal tissues. The enzymes may there-
dues linked through phosphoanhydride bonds. This fore prove to be suitable targets in the development of
polymer, present in all organisms, may accumulate to new antimicrobial drugs.
high levels in some cells. In yeast, for example, the No yeast gene encodes a PPK-like protein, but four
amount of polyP that accumulates in the vacuoles would genes—unrelated to bacterial PPK genes—are neces-
represent, if distributed uniformly throughout the cell, sary for the synthesis of polyphosphate. The mechanism
a concentration of 200 mM! (Compare this with the confor polyphosphate synthesis in eukaryotes seems to be
centrations of other phosphoryl group donors listed in quite different from that in bacteria.
Table 13–5.)
O O O O O SUMMARY 13.3 Phosphoryl Group Transfers and ATP

"O
P O P O P O P O P O ! ATP is the chemical link between catabolism and
O" O" O" O" O" anabolism. It is the energy currency of the living
Inorganic polyphosphate (polyP) cell. The exergonic conversion of ATP to ADP and
Pi, or to AMP and PPi, is coupled to many
One potential role for polyP is to serve as a phos- endergonic reactions and processes.
phagen, a reservoir of phosphoryl groups that can be ! Direct hydrolysis of ATP is the source of energy
used to generate ATP, as creatine phosphate is used in in some processes driven by conformational
muscle. PolyP has about the same phosphoryl group changes, but in general it is not ATP hydrolysis
transfer potential as PPi. The shortest polyphosphate, but the transfer of a phosphoryl, pyrophosphoryl,
PPi (n 5 2), can serve as the energy source for active or adenylyl group from ATP to a substrate or
transport of H1 across the vacuolar membrane in plant enzyme that couples the energy of ATP
cells. For at least one form of the enzyme phosphofruc- breakdown to endergonic transformations of
tokinase in plants, PPi is the phosphoryl group donor, a substrates.
role played by ATP in animals and microbes (p. 550). ! Through these group transfer reactions, ATP
The finding of high concentrations of polyP in volcanic
provides the energy for anabolic reactions,
condensates and steam vents suggests that it could
including the synthesis of informational
have served as an energy source in prebiotic and early
macromolecules, and for the transport of
cellular evolution.
molecules and ions across membranes against
In bacteria, the enzyme polyphosphate kinase-1
concentration gradients and electrical potential
(PPK-1) catalyzes the reversible reaction
gradients.
!
Mg21
ATP 1 (polyP) n 3::4 ADP 1 (polyP) n11 To maintain its high group transfer potential,
¢G¿8 5 220 kJ/mol
ATP concentration must be held far above the
equilibrium concentration by energy-yielding
by a mechanism involving an enzyme-bound P –His reactions of catabolism.
intermediate (recall the mechanism of nucleoside ! Cells contain other metabolites with large,
diphosphate kinase, described in Fig. 13–21). A second negative, free energies of hydrolysis, including
enzyme, polyphosphate kinase-2 (PPK-2), catalyzes phosphoenolpyruvate, 1,3-bisphosphoglycerate,
the reversible synthesis of GTP (or ATP) from poly- and phosphocreatine. These high-energy
phosphate and GDP (or ADP): compounds, like ATP, have a high phosphoryl
Mn21 group transfer potential. Thioesters also have
GDP 1 (polyP) n11 3::4 GTP 1 (polyP) n
high free energies of hydrolysis.
PPK-2 is believed to act primarily in the direction of ! Inorganic polyphosphate, present in all cells, may
GTP and ATP synthesis, and PPK-1 in the direction of serve as a reservoir of phosphoryl groups with high
polyphosphate synthesis. PPK-1 and PPK-2 are present group transfer potential.
13.4 Biological Oxidation-Reduction provides energy to a variety of molecular energy trans-

ducers (enzymes and other proteins) that do biological
Reactions work. In the mitochondrion, for example, membrane-
bound enzymes couple electron flow to the production of
The transfer of phosphoryl groups is a central feature of
a transmembrane pH difference and a transmembrane
metabolism. Equally important is another kind of transfer,
electrical potential, accomplishing osmotic and electrical
electron transfer in oxidation-reduction reactions. These
work. The proton gradient thus formed has potential
reactions involve the loss of electrons by one chemical
energy, sometimes called the proton-motive force by
species, which is thereby oxidized, and the gain of elec-
analogy with electromotive force. Another enzyme, ATP
trons by another, which is reduced. The flow of electrons
synthase in the inner mitochondrial membrane, uses the
in oxidation-reduction reactions is responsible, directly or
proton-motive force to do chemical work: synthesis of
indirectly, for all work done by living organisms. In non-
ATP from ADP and Pi as protons flow spontaneously
photosynthetic organisms, the sources of electrons are
across the membrane. Similarly, membrane-localized
reduced compounds (foods); in photosynthetic organ-
enzymes in E. coli convert emf to proton-motive force,
isms, the initial electron donor is a chemical species
which is then used to power flagellar motion. The prin-
excited by the absorption of light. The path of electron
ciples of electrochemistry that govern energy changes in
flow in metabolism is complex. Electrons move from
the macroscopic circuit with a motor and battery apply
various metabolic intermediates to specialized electron
with equal validity to the molecular processes accompa-
carriers in enzyme-catalyzed reactions. The carriers in
nying electron flow in living cells.
turn donate electrons to acceptors with higher electron
affinities, with the release of energy. Cells contain a
variety of molecular energy transducers, which convert Oxidation-Reductions Can Be Described
the energy of electron flow into useful work. as Half-Reactions
We begin by discussing how work can be accom- Although oxidation and reduction must occur together,
plished by an electromotive force (emf), then consider the it is convenient when describing electron transfers to
theoretical and experimental basis for measuring energy consider the two halves of an oxidation-reduction reac-
changes in oxidation reactions in terms of emf and the tion separately. For example, the oxidation of ferrous
relationship between this force, expressed in volts, and the ion by cupric ion,
free-energy change, expressed in joules. We conclude by
describing the structures and oxidation-reduction chemis- Fe21 1 Cu21 ∆ Fe31 1 Cu1
try of the most common of the specialized electron carri- can be described in terms of two half-reactions:
ers, which you will encounter repeatedly in later chapters.
(1) Fe21 ∆ Fe31 1 e 2
(2) Cu21 1 e 2 ∆ Cu 1
The Flow of Electrons Can Do Biological Work
Every time we use a motor, an electric light or heater, or a The electron-donating molecule in an oxidation-reduction
spark to ignite gasoline in a car engine, we use the flow of reaction is called the reducing agent or reductant; the
electrons to accomplish work. In the circuit that powers a electron-accepting molecule is the oxidizing agent or
motor, the source of electrons can be a battery containing oxidant. A given agent, such as an iron cation existing in
two chemical species that differ in affinity for electrons. the ferrous (Fe21) or ferric (Fe31) state, functions as a
Electrical wires provide a pathway for electron flow from conjugate reductant-oxidant pair (redox pair), just as an
the chemical species at one pole of the battery, through acid and corresponding base function as a conjugate
the motor, to the chemical species at the other pole of the acid-base pair. Recall from Chapter 2 that in acid-base
battery. Because the two chemical species differ in their reactions we can write a general equation: proton donor
affinity for electrons, electrons flow spontaneously through ∆ H1 1 proton acceptor. In redox reactions we can
the circuit, driven by a force proportional to the difference write a similar general equation: electron donor (reduc-
in electron affinity, the electromotive force, emf. The tant) ∆ e2 1 electron acceptor (oxidant). In the
emf (typically a few volts) can accomplish work if an reversible half-reaction (1) above, Fe21 is the electron
appropriate energy transducer—in this case a motor—is donor and Fe31 is the electron acceptor; together, Fe21
placed in the circuit. The motor can be coupled to a variety and Fe31 constitute a conjugate redox pair.
of mechanical devices to do useful work. The electron transfers in the oxidation-reduction
Living cells have an analogous biological “circuit,” reactions of organic compounds are not fundamentally
with a relatively reduced compound such as glucose as different from those of inorganic species. Consider the
the source of electrons. As glucose is enzymatically oxi- oxidation of a reducing sugar (an aldehyde or ketone)
dized, the released electrons flow spontaneously through by cupric ion:
a series of electron-carrier intermediates to another
chemical species, such as O2. This electron flow is exer- O O
gonic, because O2 has a higher affinity for electrons than R C ! 4OH" ! 2Cu2! R C ! Cu2O ! 2H2O
do the electron-carrier intermediates. The resulting emf H OH
13.4 Biological Oxidation-Reduction Reactions 529
This overall reaction can be expressed as two half- H

reactions: Methane H C H 8
H
O O
(1) R C ! 2OH" R C ! 2e" ! H2O
H H
H OH
Ethane H C C H 7
(alkane) H H
(2) 2Cu21 1 2e2 1 2OH2 ∆ Cu2O 1 H2O
Because two electrons are removed from the aldehyde H H

Ethene C C 6
carbon, the second half-reaction (the one-electron (alkene) H H
reduction of cupric to cuprous ion) must be doubled to
balance the overall equation. H H
Ethanol H C C O H 5
(alcohol)
Biological Oxidations Often Involve Dehydrogenation H H
The carbon in living cells exists in a range of oxidation
states (Fig. 13–22). When a carbon atom shares an Acetylene
(alkyne) H C C H 5
electron pair with another atom (typically H, C, S, N,
or O), the sharing is unequal in favor of the more elec-
H
tronegative atom. The order of increasing electronega- Formaldehyde C O 4
tivity is H , C , S , N , O. In oversimplified but H
useful terms, the more electronegative atom “owns”
the bonding electrons it shares with another atom. For H H
example, in methane (CH4), carbon is more electro- Acetaldehyde
H C C 3
(aldehyde)
negative than the four hydrogens bonded to it, and the H O
C atom therefore “owns” all eight bonding electrons
(Fig. 13–22). In ethane, the electrons in the COC
bond are shared equally, so each C atom “owns” only H O H
seven of its eight bonding electrons. In ethanol, C-1 is Acetone H C C C H 2
(ketone)
less electronegative than the oxygen to which it is H H
bonded, and the O atom therefore “owns” both elec-
trons of the COO bond, leaving C-1 with only five O
Formic acid
bonding electrons. With each formal loss of “owned” (carboxylic H C 2
electrons, the carbon atom has undergone oxidation— acid) O
H
even when no oxygen is involved, as in the conver-
sion of an alkane (OCH2OCH2O) to an alkene
Carbon C O 2
(OCHPCHO). In this case, oxidation (loss of elec- monoxide
trons) is coincident with the loss of hydrogen. In bio-
logical systems, as we noted earlier in the chapter, H O
Acetic acid
oxidation is often synonymous with dehydrogenation (carboxylic H C C 1
and many enzymes that catalyze oxidation reactions acid)
H
O
are dehydrogenases. Notice that the more reduced H
compounds in Figure 13–22 (top) are richer in hydro-
gen than in oxygen, whereas the more oxidized com- Carbon O C O 0
dioxide
pounds (bottom) have more oxygen and less hydrogen.
Not all biological oxidation-reduction reactions FIGURE 13–22 Different levels of oxidation of carbon compounds in
involve carbon. For example, in the conversion of molec- the biosphere. To approximate the level of oxidation of these com-
ular nitrogen to ammonia, 6H1 1 6e2 1 N2 n 2NH3, the pounds, focus on the red carbon atom and its bonding electrons. When
nitrogen atoms are reduced. this carbon is bonded to the less electronegative H atom, both bonding
Electrons are transferred from one molecule (elec- electrons (red) are assigned to the carbon. When carbon is bonded to
tron donor) to another (electron acceptor) in one of another carbon, bonding electrons are shared equally, so one of the two
four ways: electrons is assigned to the red carbon. When the red carbon is bonded
to the more electronegative O atom, the bonding electrons are assigned
1. Directly as electrons. For example, the Fe21/Fe31 to the oxygen. The number to the right of each compound is the number
redox pair can transfer an electron to the Cu1/Cu21 of electrons “owned” by the red carbon, a rough expression of the
redox pair: degree of oxidation of that compound. As the red carbon undergoes
oxidation (loses electrons), the number gets smaller.
Fe21 1 Cu21 ∆ Fe31 1 Cu1
2. As hydrogen atoms. Recall that a hydrogen atom Device for

consists of a proton (H1) and a single electron measuring emf
(e'). In this case we can write the general ! "
equation
AH2 ∆ A 1 2e2 1 2H1
where AH2 is the hydrogen/electron donor. (Do

not mistake the above reaction for an acid H2 gas
dissociation, which involves a proton and no (standard Salt bridge
pressure) (KCl solution)
electron.) AH2 and A together constitute a
conjugate redox pair (A/AH2), which can reduce
another compound B (or redox pair, B/BH2) by
transfer of hydrogen atoms:
AH2 1 B ∆ A 1 BH2
3. As a hydride ion (:H2), which has two electrons.

This occurs in the case of NAD-linked
dehydrogenases, described below.
4. Through direct combination with oxygen. In this
case, oxygen combines with an organic reductant Reference cell of known emf: Test cell containing 1 M
the hydrogen electrode in concentrations of the
and is covalently incorporated in the product, as in oxidized and reduced
which H2 gas at 101.3 kPa is
the oxidation of a hydrocarbon to an alcohol: equilibrated at the electrode species of the redox pair
with 1 M H" to be examined
ROCH3 1 12 O2 88n ROCH2OOH
The hydrocarbon is the electron donor and the FIGURE 13–23 Measurement of the standard reduction potential (E!")
oxygen atom is the electron acceptor. of a redox pair. Electrons flow from the test electrode to the reference
electrode, or vice versa. The ultimate reference half-cell is the hydrogen
All four types of electron transfer occur in cells. The electrode, as shown here, at pH 0. The electromotive force (emf) of this
neutral term reducing equivalent is commonly used electrode is designated 0.00 V. At pH 7 in the test cell (and 25 8C), E98
to designate a single electron equivalent participating in for the hydrogen electrode is 20.414 V. The direction of electron flow
an oxidation-reduction reaction, no matter whether this depends on the relative electron “pressure” or potential of the two cells.
equivalent is an electron per se or part of a hydrogen A salt bridge containing a saturated KCl solution provides a path for
atom or a hydride ion, or whether the electron transfer counter-ion movement between the test cell and the reference cell.
takes place in a reaction with oxygen to yield an oxy- From the observed emf and the known emf of the reference cell, the
genated product. Because biological fuel molecules are experimenter can find the emf of the test cell containing the redox pair.
usually enzymatically dehydrogenated to lose two The cell that gains electrons has, by convention, the more positive
reducing equivalents at a time, and because each oxy- reduction potential.
gen atom can accept two reducing equivalents, bio-
chemists by convention regard the unit of biological
oxidations as two reducing equivalents passing from
substrate to oxygen. When this hydrogen electrode is connected through
an external circuit to another half-cell in which an
oxidized species and its corresponding reduced spe-
Reduction Potentials Measure Affinity for Electrons cies are present at standard concentrations (25 8C,
When two conjugate redox pairs are together in solu- each solute at 1 M , each gas at 101.3 kPa), electrons
tion, electron transfer from the electron donor of one tend to flow through the external circuit from the
pair to the electron acceptor of the other may proceed half-cell of lower E8 to the half-cell of higher E8. By
spontaneously. The tendency for such a reaction convention, a half-cell that takes electrons from the
depends on the relative affinity of the electron acceptor standard hydrogen cell is assigned a positive value
of each redox pair for electrons. The standard reduc- of E8, and one that donates electrons to the hydro-
tion potential, E8, a measure (in volts) of this affinity, gen cell, a negative value. When any two half-cells
can be determined in an experiment such as that are connected, that with the larger (more positive)
described in Figure 13–23. Electrochemists have cho- E8 will get reduced; it has the greater reduction
sen as a standard of reference the half-reaction potential.
The reduction potential of a half-cell depends not
H1 1 e2 88n 12 H2
only on the chemical species present but also on their
The electrode at which this half-reaction occurs activities, approximated by their concentrations. About
(called a half-cell) is arbitrarily assigned an E8 of 0.00 V. a century ago, Walther Nernst derived an equation that
relates standard reduction potential (E8) to the actual

reduction potential (E) at any concentration of oxidized TABLE 13–7 Standard Reduction Potentials of Some
and reduced species in a living cell: Biologically Important Half-Reactions
RT [electron acceptor] Half-reaction E98 (V)
E 5 E8 1 ln (13–5)
n [electron donor 4 1 1 2
2 O2 1 2H 1 2e 88n H2O 0.816
where R and T have their usual meanings, n is the num-
Fe31 1 e2 88n Fe21 0.771
ber of electrons transferred per molecule, and is the
Faraday constant (Table 13–1). At 298 K (25 8C), this NO2 1 2 2
3 1 2H 1 2e 88n NO2 1 H2O 0.421
expression reduces to Cytochrome f (Fe31 ) 1 e2 88n
cytochrome f (Fe21 ) 0.365
0.026V [electron acceptor 4
E 5 E8 1 ln (13–6) Fe(CN) 32 (ferricyanide) 1 e2 88n Fe(CN) 42 0.36
n [electron donor 4 6 6
Cytochrome a3 (Fe31 ) 1 e2 88n

KEY CONVENTION: Many half-reactions of interest to bio- cytochrome a3 (Fe21 ) 0.35
chemists involve protons. As in the definition of DG98, O2 1 2H1 1 2e2 88n H2O2 0.295
biochemists define the standard state for oxidation-
Cytochrome a (Fe31 ) 1 e2 88n
reduction reactions as pH 7 and express a standard
cytochrome a (Fe21 ) 0.29
transformed reduction potential, E98, the standard
reduction potential at pH 7 and 25 8C. By convention, Cytochrome c (Fe31 ) 1 e2 88n
DE98 for any redox reaction is given as E98 of the elec- cytochrome c (Fe21 ) 0.254
tron acceptor minus E98 of the electron donor. ■ Cytochrome c1 (Fe31 ) 1 e2 88n
cytochrome c1 (Fe21 ) 0.22
The standard reduction potentials given in Table Cytochrome b (Fe31 ) 1 e2 88n
13–7 and used throughout this book are values for E98 cytochrome b (Fe21 ) 0.077
and are therefore valid only for systems at neutral
Ubiquinone 1 2H1 1 2e2 88n ubiquinol 1 H2 0.045
pH. Each value represents the potential difference
when the conjugate redox pair, at 1 M concentrations, Fumarate22 1 2H1 1 2e2 88n succinate22 0.031
25 8C, and pH 7, is connected with the standard (pH 2H1 1 2e2 88n H2 (at standard conditions, pH 0) 0.000
0) hydrogen electrode. Notice in Table 13–7 that Crotonyl-CoA 1 2H1 1 2e2 88n butyryl-CoA 20.015
when the conjugate pair 2H1/H2 at pH 7 is connected
Oxaloacetate22 1 2H1 1 2e2 88n malate22 20.166
with the standard hydrogen electrode (pH 0), elec-
trons tend to flow from the pH 7 cell to the standard Pyruvate2 1 2H1 1 2e2 88n lactate2 20.185
(pH 0) cell; the measured E98 for the 2H1/H2 pair is Acetaldehyde 1 2H1 1 2e2 88n ethanol 20.197
20.414 V. FAD 1 2H1 1 2e2 88n FADH2 20.219*
Glutathione 1 2H1 1 2e2 88n
Standard Reduction Potentials Can Be Used 2 reduced glutathione 20.23
to Calculate Free-Energy Change S 1 2H1 1 2e2 88n H2S 20.243
Why are reduction potentials so useful to the biochemist? Lipoic acid 1 2H1 1 2e2 88n dihydrolipoic acid 20.29
When E values have been determined for any two half- NAD1 1 H1 1 2e2 88n NADH 20.320
cells, relative to the standard hydrogen electrode, we
NADP1 1 H1 1 2e2 88n NADPH 20.324
also know their reduction potentials relative to each
other. We can then predict the direction in which elec- Acetoacetate 1 2H1 1 2e2 88n
trons will tend to flow when the two half-cells are con- %-hydroxybutyrate 20.346
nected through an external circuit or when components $-Ketoglutarate 1 CO2 1 2H1 1 2e2 88n
of both half-cells are present in the same solution. Elec- isocitrate 20.38
trons tend to flow to the half-cell with the more positive 1 2
2H 1 2e 88n H2 (at pH 7) 20.414
E, and the strength of that tendency is proportional to
Ferredoxin (Fe31 ) 1 e2 88n ferredoxin (Fe21 ) 20.432
DE, the difference in reduction potential. The energy
made available by this spontaneous electron flow (the Source: Data mostly from Loach, R.A. (1976) in Handbook of Biochemistry and Molecular
free-energy change, DG, for the oxidation-reduction Biology, 3rd edn (Fasman, G.D., ed.), Physical and Chemical Data, Vol. 1, pp. 122–130, CRC
Press, Boca Raton, FL.
reaction) is proportional to DE:
* This is the value for free FAD; FAD bound to a specific flavoprotein (e.g., succinate
¢G 5 2n ¢E or ¢G¿8 5 2n ¢E¿8 (13–7) dehydrogenase) has a different E98 that depends on its protein environment.
where n is the number of electrons transferred in the reaction from the values of E98 in a table of reduction
reaction. With this equation we can calculate the potentials (Table 13–7) and the concentrations of
actual free-energy change for any oxidation-reduction reacting species.
Cells convert glucose to CO2 not in a single, high-ener-

WORKED EXAMPLE 13–3 Calculation of DG98 and DG of gy-releasing reaction but rather in a series of controlled
a Redox Reaction reactions, some of which are oxidations. The free
Calculate the standard free-energy change, DG98, for energy released in these oxidation steps is of the same
the reaction in which acetaldehyde is reduced by the order of magnitude as that required for ATP synthesis
biological electron carrier NADH: from ADP, with some energy to spare. Electrons
removed in these oxidation steps are transferred to
Acetaldehyde 1 NADH 1 H1 88n ethanol 1 NAD1 coenzymes specialized for carrying electrons, such as
Then calculate the actual free-energy change, DG, NAD1 and FAD (described below).
when [acetaldehyde] and [NADH] are 1.00 M, and [etha-
nol] and [NAD1] are 0.100 M. The relevant half-reactions A Few Types of Coenzymes and Proteins Serve
and their E98 values are:
as Universal Electron Carriers
(1) Acetaldehyde 1 2H1 1 2e2 ¡ ethanol The multitude of enzymes that catalyze cellular oxida-
E98 5 20.197 V tions channel electrons from their hundreds of different
(2) NAD1 1 2H1 1 2e2 ¡ NADH 1 H1 substrates into just a few types of universal electron car-
E98 5 20.320 V riers. The reduction of these carriers in catabolic pro-
cesses results in the conservation of free energy released
Remember that, by convention, DE98 is E98 of the elec- by substrate oxidation. NAD, NADP, FMN, and FAD are
tron acceptor minus E98 of the electron donor. water-soluble coenzymes that undergo reversible oxida-
tion and reduction in many of the electron-transfer reac-
Solution: Because acetaldehyde is accepting electrons
tions of metabolism. The nucleotides NAD and NADP
(n 5 2) from NADH, DE98 5 20.197 V 2 (20.320 V) 5
move readily from one enzyme to another; the flavin
0.123 V. Therefore,
nucleotides FMN and FAD are usually very tightly bound
DG98 5 2 n DE98 5 22(96.5 kJ/V ? mol)(0.123 V) to the enzymes, called flavoproteins, for which they
5 223.7 kJ/mol serve as prosthetic groups. Lipid-soluble quinones such
as ubiquinone and plastoquinone act as electron carriers
This is the free-energy change for the oxidation-reduction
and proton donors in the nonaqueous environment of
reaction at 25 8C and pH 7, when acetaldehyde, ethanol,
membranes. Iron-sulfur proteins and cytochromes, which
NAD1, and NADH are all present at 1.00 M concentrations.
have tightly bound prosthetic groups that undergo revers-
To calculate DG when [acetaldehyde] and [NADH]
ible oxidation and reduction, also serve as electron car-
are 1.00 M, and [ethanol] and [NAD1] are 0.100 M, we can
riers in many oxidation-reduction reactions. Some of
use Equation 13–4 and the standard free-energy change
these proteins are water-soluble, but others are periph-
we calculated above:
eral or integral membrane proteins (see Fig. 11–7).
[ethanol] [NAD 1 ] We conclude this chapter by describing some chemi-
DG 5 DG98 1 RT In
[acetaldehyde ] [NADH ] cal features of nucleotide coenzymes and some of the
5 223.7 kJ/mol 1 enzymes (dehydrogenases and flavoproteins) that use
(0.100 M )(0.100 M ) them. The oxidation-reduction chemistry of quinones,
(8.315 J/mol ?K)(298 K) ln
(1.00 M ) (1.00 M ) iron-sulfur proteins, and cytochromes is discussed in
5 223.7 kJ/mol 1 (2.48 J/mol) ln 0.01 Chapter 19.
5 235.1 kJ/mol
This is the actual free-energy change at the specified NADH and NADPH Act with Dehydrogenases
concentrations of the redox pairs. as Soluble Electron Carriers
Nicotinamide adenine dinucleotide (NAD; NAD1 in its
oxidized form) and its close analog nicotinamide ade-
Cellular Oxidation of Glucose to Carbon Dioxide nine dinucleotide phosphate (NADP; NADP1 when
Requires Specialized Electron Carriers oxidized) are composed of two nucleotides joined
The principles of oxidation-reduction energetics described through their phosphate groups by a phosphoanhydride
above apply to the many metabolic reactions that involve bond (Fig. 13–24a). Because the nicotinamide ring
electron transfers. For example, in many organisms, the resembles pyridine, these compounds are sometimes
oxidation of glucose supplies energy for the production of called pyridine nucleotides. The vitamin niacin is the
ATP. The complete oxidation of glucose: source of the nicotinamide moiety in nicotinamide
nucleotides.
C6H12O6 1 6O2 88n 6CO2 1 6H2O
Both coenzymes undergo reversible reduction of the
has a DG98 of 22,840 kJ/mol. This is a much larger nicotinamide ring (Fig. 13–24). As a substrate molecule
release of free energy than is required for ATP synthesis undergoes oxidation (dehydrogenation), giving up two
in cells (50 to 60 kJ/mol; see Worked Example 13–2). hydrogen atoms, the oxidized form of the nucleotide
O O O
H B H H B H H B
C 2e!
? C ? CH
H H
NH2 NH2 or NH2 " H"
"
N 2H "
N N
O CH2 O A A
R A side R B side
H H
OP PO O! H H NADH
(reduced)
O OH OH
NH2
N
OP PO O ! N Adenine
1.0
N Oxidized
O CH2 O N (NAD")
0.8
Absorbance
H H
H H 0.6
NAD! Reduced
OH OH 0.4
(oxidized) (NADH)
In NADP" this hydroxyl group 0.2
is esterified with phosphate.
0.0
(a) 220 240 260 280 300 320 340 360 380
Wavelength (nm)
(b)
FIGURE 13–24 NAD and NADP. (a) Nicotinamide adenine dinucleotide, NADH. Reduction of the nicotinamide ring produces a new, broad
NAD1, and its phosphorylated analog NADP1 undergo reduction to absorption band with a maximum at 340 nm. The production of NADH
NADH and NADPH, accepting a hydride ion (two electrons and one during an enzyme-catalyzed reaction can be conveniently followed by
proton) from an oxidizable substrate. The hydride ion is added to either observing the appearance of the absorbance at 340 nm (molar extinc-
the front (the A side) or the back (the B side) of the planar nicotinamide tion coefficient e340 5 6,200 M21 cm21).
ring (see Table 13–8). (b) The UV absorption spectra of NAD1 and
(NAD1 or NADP1) accepts a hydride ion (:H2, the NAD1 (oxidized) to NADH (reduced) is high, favoring
equivalent of a proton and two electrons) and is reduced hydride transfer from a substrate to NAD1 to form NADH.
(to NADH or NADPH). The second proton removed By contrast, NADPH is generally present at a higher con-
from the substrate is released to the aqueous solvent. centration than NADP1, favoring hydride transfer from
The half-reactions for these nucleotide cofactors are NADPH to a substrate. This reflects the specialized meta-
bolic roles of the two coenzymes: NAD1 generally func-
NAD1 1 2e2 1 2H1 ¡ NADH 1 H1 tions in oxidations—usually as part of a catabolic reaction;
NADP1 1 2e2 1 2H1 ¡ NADPH 1 H1 NADPH is the usual coenzyme in reductions—nearly
always as part of an anabolic reaction. A few enzymes can
Reduction of NAD1 or NADP1 converts the benzenoid use either coenzyme, but most show a strong preference
ring of the nicotinamide moiety (with a fixed positive for one over the other. Also, the processes in which these
charge on the ring nitrogen) to the quinonoid form (with two cofactors function are segregated in eukaryotic cells:
no charge on the nitrogen). The reduced nucleotides for example, oxidations of fuels such as pyruvate, fatty
absorb light at 340 nm; the oxidized forms do not (Fig. acids, and $-keto acids derived from amino acids occur in
13–24b); this difference in absorption is used by bio- the mitochondrial matrix, whereas reductive biosynthetic
chemists to assay reactions involving these coenzymes. processes such as fatty acid synthesis take place in the
Note that the plus sign in the abbreviations NAD1 and cytosol. This functional and spatial specialization allows a
NADP1 does not indicate the net charge on these mol- cell to maintain two distinct pools of electron carriers,
ecules (in fact, both are negatively charged); rather, it with two distinct functions.
indicates that the nicotinamide ring is in its oxidized More than 200 enzymes are known to catalyze reac-
form, with a positive charge on the nitrogen atom. In the tions in which NAD1 (or NADP1) accepts a hydride ion
abbreviations NADH and NADPH, the “H” denotes the from a reduced substrate, or NADPH (or NADH)
added hydride ion. To refer to these nucleotides without donates a hydride ion to an oxidized substrate. The
specifying their oxidation state, we use NAD and NADP. general reactions are
The total concentration of NAD1 1 NADH in most
tissues is about 1025 M; that of NADP1 1 NADPH is AH2 1 NAD1 ¡ A 1 NADH 1 H1
about 1026 M. In many cells and tissues, the ratio of A 1 NADPH 1 H1 ¡ AH2 1 NADP1
where AH2 is the reduced substrate and A the oxidized (a)

substrate. The general name for an enzyme of this type
is oxidoreductase; they are also commonly called
dehydrogenases. For example, alcohol dehydrogenase
catalyzes the first step in the catabolism of ethanol, in
which ethanol is oxidized to acetaldehyde:
CH3CH2OH 1 NAD1 ¡ CH3CHO 1 NADH 1 H1
Ethanol Acetaldehyde
Notice that one of the carbon atoms in ethanol has lost

a hydrogen; the compound has been oxidized from an
alcohol to an aldehyde (refer again to Fig. 13–22 for the
oxidation states of carbon). (b)
When NAD1 or NADP1 is reduced, the hydride
ion could in principle be transferred to either side of
the nicotinamide ring: the front (A side) or the back NAD
(B side), as represented in Figure 13–24a. Studies with
isotopically labeled substrates have shown that a given
enzyme catalyzes either an A-type or a B-type transfer,
but not both. For example, yeast alcohol dehydrogenase
and lactate dehydrogenase of vertebrate heart transfer
a hydride ion to (or remove a hydride ion from) the A
side of the nicotinamide ring; they are classed as type A
dehydrogenases to distinguish them from another group
of enzymes that transfer a hydride ion to (or remove a
hydride ion from) the B side of the nicotinamide ring
Rossmann fold 1 Rossmann fold 2
(Table 13–8). The specificity for one side or another can
be very striking; lactate dehydrogenase, for example,
FIGURE 13–25 The Rossmann fold. This structural motif is found in the
prefers the A side over the B side by a factor of 5 3 107!
NAD-binding site of many dehydrogenases. (a) It consists of a pair of
The basis for this preference lies in the exact position-
structurally similar motifs (only one of which is shown here), each having
ing of the enzyme groups involved in hydrogen bonding
three parallel % sheets and two $ helices (%-$-%-$-%). (b) The nucleo-
with the OCONH2 group of the nicotinamide. tide-binding domain of the enzyme lactate dehydrogenase (derived from
Most dehydrogenases that use NAD or NADP bind PDB ID 3LDH) with NAD (ball-and-stick structure) bound in an extended
the cofactor in a conserved protein domain called the conformation through hydrogen bonds and salt bridges to the paired
Rossmann fold (named for Michael Rossmann, who %-$-%-$-% motifs of the Rossmann fold (shades of red and blue).
deduced the structure of lactate dehydrogenase and
first described this structural motif). The Rossmann The association between a dehydrogenase and NAD
fold typically consists of a six-stranded parallel % sheet or NADP is relatively loose; the coenzyme readily diffuses
and four associated $ helices (Fig. 13–25). from one enzyme to another, acting as a water-soluble
TABLE 13–8 Stereospecificity of Dehydrogenases That Employ NAD1 or NADP1 as Coenzymes

Stereochemical
specificity for
nicotinamide
Enzyme Coenzyme ring (A or B) Text page
1
Isocitrate dehydrogenase NAD A 643
$-Ketoglutarate dehydrogenase NAD1 B 644
Glucose 6-phosphate dehydrogenase NADP1 B 577
Malate dehydrogenase NAD1 A 647
Glutamate dehydrogenase NAD1 or NADP1 B 702
Glyceraldehyde 3-phosphate dehydrogenase NAD1 B 553
Lactate dehydrogenase NAD1 A 563
Alcohol dehydrogenase NAD1 A 565
carrier of electrons from one metabolite to another. For acterized by the “three Ds”:
example, in the production of alcohol during fermenta- dermatitis, diarrhea, and
tion of glucose by yeast cells, a hydride ion is removed dementia, followed in many
from glyceraldehyde 3-phosphate by one enzyme (glyc- cases by death. A century ago,
eraldehyde 3-phosphate dehydrogenase, a type B pellagra was a common human
enzyme) and transferred to NAD1. The NADH pro- disease; in the southern United
duced then leaves the enzyme surface and diffuses to States, where maize was a
another enzyme (alcohol dehydrogenase, a type A dietary staple, about 100,000
enzyme), which transfers a hydride ion to acetaldehyde, people were afflicted and about
producing ethanol: 10,000 died as a result of this
disease between 1912 and
(1) Glyceraldehyde 3-phosphate 1 NAD1 ¡
1916. In 1920 Joseph Gold- Frank Strong,
3-phosphoglycerate 1 NADH 1 H1
berger showed pellagra to be 1908–1993
(2) Acetaldehyde 1 NADH 1 H1 ¡ ethanol 1 NAD1
caused by a dietary insuffi-
Sum: Glyceraldehyde 3-phosphate 1 acetaldehyde ¡ ciency, and in 1937 Frank
3-phosphoglycerate 1 ethanol Strong, D. Wayne Woolley, and
Notice that in the overall reaction there is no net produc- Conrad Elvehjem identified
tion or consumption of NAD1 or NADH; the coenzymes niacin as the curative agent for
function catalytically and are recycled repeatedly with- blacktongue. Supplementation
out a net change in the concentration of NAD1 1 NADH. of the human diet with this
inexpensive compound has
Dietary Deficiency of Niacin, the Vitamin Form eradicated pellagra in the
populations of the developed
of NAD and NADP, Causes Pellagra world, with one significant
As we noted in Chapter 6, and will discuss fur- exception: people with alco- D. Wayne Woolley,
ther in the chapters to follow, most coenzymes holism, or who drink excessive 1914–1966
are derived from the substances we call vitamins. The amounts of alcohol. In these
pyridine-like rings of NAD and NADP are derived from individuals, intestinal absorp-
the vitamin niacin (nicotinic acid; Fig. 13–26), which tion of niacin is much reduced,
is synthesized from tryptophan. Humans generally can- and caloric needs are often
not synthesize sufficient quantities of niacin, and this is met with distilled spirits that
especially so for individuals with diets low in trypto- are virtually devoid of vita-
phan (maize, for example, has a low tryptophan con- mins, including niacin. In
tent). Niacin deficiency, which affects all the NAD(P)- some parts of the world, includ-
dependent dehydrogenases, causes the serious human ing the Deccan Plateau in
disease pellagra (Italian for “rough skin”) and a related India, pellagra still occurs in
disease in dogs, blacktongue. These diseases are char- the general population, espe-
cially among people living in Conrad Elvehjem,
poverty. ■ 1901–1962
O
C O"
(
Flavin Nucleotides Are Tightly Bound in Flavoproteins
Flavoproteins (Table 13–9) are enzymes that catalyze
( ( CH3 oxidation-reduction reactions using either flavin mono-
Niacin Nicotine nucleotide (FMN) or flavin adenine dinucleotide (FAD)
(nicotinic acid)
as coenzyme (Fig. 13–27). These coenzymes, the
O NH3
! flavin nucleotides, are derived from the vitamin ribo-
flavin. The fused ring structure of flavin nucleotides
C CH2 CH COO" (the isoalloxazine ring) undergoes reversible reduction,
NH2
accepting either one or two electrons in the form of one
( ( or two hydrogen atoms (each atom an electron plus a
) proton) from a reduced substrate. The fully reduced
Nicotinamide Tryptophan
forms are abbreviated FADH2 and FMNH2. When a fully
FIGURE 13–26 Niacin (nicotinic acid) and its derivative nicotinamide. oxidized flavin nucleotide accepts only one electron
The biosynthetic precursor of these compounds is tryptophan. In the labo- (one hydrogen atom), the semiquinone form of the iso-
ratory, nicotinic acid was first produced by oxidation of the natural product alloxazine ring is produced, abbreviated FADH• and
nicotine—thus the name. Both nicotinic acid and nicotinamide cure pella- FMNH•. Because flavin nucleotides have a slightly dif-
gra, but nicotine (from cigarettes or elsewhere) has no curative activity. ferent chemical specialty from that of the nicotinamide
The flavin nucleotide in most flavoproteins is bound

TABLE 13–9 Some Enzymes (Flavoproteins) That rather tightly to the protein, and in some enzymes, such
Employ Flavin Nucleotide Coenzymes as succinate dehydrogenase, it is bound covalently. Such
Flavin tightly bound coenzymes are properly called prosthetic
Enzyme nucleotide Text page(s) groups. They do not transfer electrons by diffusing from
one enzyme to another; rather, they provide a means by
Acyl-CoA dehydrogenase FAD 673 which the flavoprotein can temporarily hold electrons
Dihydrolipoyl dehydrogenase FAD 637 while it catalyzes electron transfer from a reduced sub-
Succinate dehydrogenase FAD 646 strate to an electron acceptor. One important feature of
the flavoproteins is the variability in the standard reduc-
Glycerol 3-phosphate
tion potential (E98) of the bound flavin nucleotide. Tight
dehydrogenase FAD 759
association between the enzyme and prosthetic group
Thioredoxin reductase FAD 917 confers on the flavin ring a reduction potential typical of
NADH dehydrogenase that particular flavoprotein, sometimes quite different
(Complex I) FMN 738–739 from the reduction potential of the free flavin nucleo-
tide. FAD bound to succinate dehydrogenase, for exam-
Glycolate oxidase FMN 813
ple, has an E98 close to 0.0 V, compared with 20.219 V
for free FAD; E98 for other flavoproteins ranges from
20.40 V to 1 0.06 V. Flavoproteins are often very com-
coenzymes—the ability to participate in either one- or plex; some have, in addition to a flavin nucleotide,
two-electron transfers—flavoproteins are involved in a tightly bound inorganic ions (iron or molybdenum, for
greater diversity of reactions than the NAD(P)-linked example) capable of participating in electron transfers.
dehydrogenases. Certain flavoproteins act in a quite different role, as
Like the nicotinamide coenzymes (Fig. 13–24), the light receptors. Cryptochromes are a family of flavopro-
flavin nucleotides undergo a shift in a major absorption teins, widely distributed in the eukaryotic phyla, that
band on reduction (again, useful to biochemists who want mediate the effects of blue light on plant development and
to monitor reactions involving these coenzymes). Flavo- the effects of light on mammalian circadian rhythms (oscil-
proteins that are fully reduced (two electrons accepted) lations in physiology and biochemistry, with a 24-hour
generally have an absorption maximum near 360 nm. period). The cryptochromes are homologs of another family
When partially reduced (one electron), they acquire of flavoproteins, the photolyases. Found in both bacteria
another absorption maximum at about 450 nm; when fully and eukaryotes, photolyases use the energy of absorbed
oxidized, the flavin has maxima at 370 and 440 nm. light to repair chemical defects in DNA.
isoalloxazine ring
O H O! H O
CH3 N H %e% ! CH3 N
%
H% % e! CH3 N
NH • NH NH
CH3 N N O CH3 N N O CH3 N N O

CH2 R R H
HCOH FADH• (FMNH•) FADH2 (FMNH2)
FMN (semiquinone) (fully reduced)
HCOH
HCOH
CH2
FAD
O
!
O P O
O
NH2
!
O P O N
N
O
N N
CH2 O
FIGURE 13–27 Oxidized and reduced FAD and FMN. FMN consists of
H H the structure above the dashed line on the FAD (oxidized form). The fla-
H H
vin nucleotides accept two hydrogen atoms (two electrons and two pro-
OH OH tons), both of which appear in the flavin ring system. When FAD or
Flavin adenine dinucleotide (FAD) and FMN accepts only one hydrogen atom, the semiquinone, a stable free
flavin mononucleotide (FMN) radical, forms.
Further Reading 537
We examine the function of flavoproteins as elec- carbanion 512 polyphosphate kinase-1,

tron carriers in Chapter 19, when we consider their carbocation 512 kinase-2 527
roles in oxidative phosphorylation (in mitochondria) aldol condensation 513 electromotive force
and photophosphorylation (in chloroplasts), and we Claisen condensation 513 (emf) 528
describe the photolyase reactions in Chapter 25. kinases 516 conjugate redox pair 528
phosphorylation potential dehydrogenation 529
SUMMARY 13.4 Biological Oxidation-Reduction (DGp) 518 dehydrogenases 529
thioester 521 reducing equivalent 530
Reactions
adenylylation 524 standard reduction
! In many organisms, a central energy-conserving inorganic potential (E98) 530
process is the stepwise oxidation of glucose to pyrophosphatase 524 pyridine nucleotide 532
CO2, in which some of the energy of oxidation is nucleoside diphosphate oxidoreductase 534
conserved in ATP as electrons are passed to O2. kinase 526 flavoprotein 535
! Biological oxidation-reduction reactions can be adenylate kinase 526 flavin nucleotides 535
described in terms of two half-reactions, each with creatine kinase 526 cryptochrome 536
a characteristic standard reduction potential, E98. phosphagens 527 photolyase 536
! When two electrochemical half-cells, each
containing the components of a half-reaction, are
connected, electrons tend to flow to the half-cell Further Reading
with the higher reduction potential. The strength Bioenergetics and Thermodynamics
of this tendency is proportional to the difference Atkins, P.W. (1984) The Second Law, Scientific American Books,
between the two reduction potentials (DE) and is Inc., New York.
a function of the concentrations of oxidized and A well-illustrated and elementary discussion of the second law
reduced species. and its implications.
!
Atkinson, D.E. (1977) Cellular Energy Metabolism and Its
The standard free-energy change for an oxidation-
Regulation, Academic Press, Inc., New York.
reduction reaction is directly proportional to the A classic treatment of the roles of ATP, ADP, and AMP in
difference in standard reduction potentials of the controlling the rate of catabolism.
two half-cells: DG98 5 2n DE98. Bergethon, P.R. (1998) The Physical Basis of Biochemistry,
! Many biological oxidation reactions are Springer Verlag, New York.
Chapters 11 through 13 of this book, and the books by Tinoco
dehydrogenations in which one or two hydrogen et al. and van Holde et al. (below), are excellent general references
atoms (H1 1 e2) are transferred from a substrate for physical biochemistry, with good discussions of the applications
to a hydrogen acceptor. Oxidation-reduction of thermodynamics to biochemistry.
reactions in living cells involve specialized electron Edsall, J.T. & Gutfreund, H. (1983) Biothermodynamics: The
carriers. Study of Biochemical Processes at Equilibrium, John Wiley &
Sons, Inc., New York.
! NAD and NADP are the freely diffusible
Hammes, G. (2000) Thermodynamics and Kinetics for the
coenzymes of many dehydrogenases. Both NAD1 Biological Sciences, John Wiley & Sons, Inc., New York.
and NADP1 accept two electrons and one proton. Clearly written, well illustrated, with excellent examples and
! FAD and FMN, the flavin nucleotides, serve as problems.
tightly bound prosthetic groups of flavoproteins. Harold, F.M. (1986) The Vital Force: A Study of Bioenergetics,
W.H. Freeman and Company, New York.
They can accept either one or two electrons and
A beautifully clear discussion of thermodynamics in biological
one or two protons. Flavoproteins also serve as processes.
light receptors in cryptochromes and photolyases. Harris, D.A. (1995) Bioenergetics at a Glance, Blackwell Science,
Oxford.
A short, clearly written account of cellular energetics, including
introductory chapters on thermodynamics.
Key Terms Haynie, D.T. (2001) Biological Thermodynamics, Cambridge

University Press, Cambridge.
Highly accessible discussions of thermodynamics and kinetics in
Terms in bold are defined in the glossary.
biological systems.
autotroph 501 anabolism 502 Loewenstein, W.R. (1999) The Touchstone of Life: Molecular
heterotroph 501 standard transformed Information, Cell Communication, and the Foundations of Life,
metabolism 502 constants 507 Oxford University Press, New York.
Beautifully written discussion of the relationship between
metabolic pathways 502 homolytic cleavage 512
entropy and information.
metabolite 502 radical 512
Nicholls, D.G. & Ferguson, S.J. (2002) Bioenergetics 3,
intermediary heterolytic cleavage 512 Academic Press, Inc., New York.
metabolism 502 nucleophile 512 Clear, well-illustrated, intermediate-level discussion of the theory
catabolism 502 electrophile 512 of bioenergetics and the mechanisms of energy transductions.

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c13BioenergeticsAndBiochemicalReactionTypes.

indd Page 505 16/08/12 8:12 AM user-F408 /Users/user-F408/Desktop

506 Bioenergetics and Biochemical Reaction Types

The units of DG and DH are joules/mole or calories/mole

13.1 Bioenergetics and Thermodynamics 507

concentration of reactants and products, the rates of

508 Bioenergetics and Biochemical Reaction Types

Chapter 6, there is a simple relationship between K¿eq

13.1 Bioenergetics and Thermodynamics 509

TABLE 13–4 Standard Free-Energy Changes of Some Chemical Reactions

510 Bioenergetics and Biochemical Reaction Types

13.2 Chemical Logic and Common Biochemical Reactions 511

512 Bioenergetics and Biochemical Reaction Types

recognize the plausible reactions can be a great aid in Nucleophiles Electrophiles

13.2 Chemical Logic and Common Biochemical Reactions 513

and carbocations are generally so unstable that their O R2 R3 O R2 R3

514 Bioenergetics and Biochemical Reaction Types

CH3 fructose 6-phosphate from glucose 6-phosphate in gly-

H affect overall oxidation state is the loss of water from an

O O Similar reactions can result from eliminations in amines.

(a) H OH H H H O" H OH H H H O"

13.2 Chemical Logic and Common Biochemical Reactions 515

and some rearrangement reactions, such as that cata- (a)

a metabolic intermediate to “activate” the intermediate ADP Glucose 6-phosphate,

516 Bioenergetics and Biochemical Reaction Types

but inaccurate picture. In Pi, four equivalent phospho- OH 2H! ! 2e" O

13.3 Phosphoryl Group Transfers and ATP 517

518 Bioenergetics and Biochemical Reaction Types

TABLE 13–5 Adenine Nucleotide, Inorganic Phosphate, and

13.3 Phosphoryl Group Transfers and ATP 519

WORKED EXAMPLE 13–2 Calculation of DGp

520 Bioenergetics and Biochemical Reaction Types

FIGURE 13–13 Hydrolysis of phosphoenolpyru- O "

PEP3" ! H2O pyruvate" ! HPO2"

$G%& # "49.3 kJ/mol

COO" COO" COO"

nance stabilized. $G%& # "43.0 kJ/mol

13.3 Phosphoryl Group Transfers and ATP 521

Thioester Extra stabilization of

J FIGURE 13–17 Free energy of hydrolysis for

522 Bioenergetics and Biochemical Reaction Types

13.3 Phosphoryl Group Transfers and ATP 523

524 Bioenergetics and Biochemical Reaction Types

13.3 Phosphoryl Group Transfers and ATP 525

BOX 13–1 Firefly Flashes: Glowing Reports of ATP

The firefly, a beetle of the Lampyridae family. S S

FIGURE 1 Important components in the firefly bioluminescence cycle.

526 Bioenergetics and Biochemical Reaction Types

Adenosine P P P Enz His Nucleoside P P P

Adenosine P P Enz His P Nucleoside P P

13.3 Phosphoryl Group Transfers and ATP 527

O O O O O SUMMARY 13.3 Phosphoryl Group Transfers and ATP

528 Bioenergetics and Biochemical Reaction Types

13.4 Biological Oxidation-Reduction provides energy to a variety of molecular energy trans-

13.4 Biological Oxidation-Reduction Reactions 529

This overall reaction can be expressed as two half- H

Because two electrons are removed from the aldehyde H H

530 Bioenergetics and Biochemical Reaction Types

2. As hydrogen atoms. Recall that a hydrogen atom Device for

where AH2 is the hydrogen/electron donor. (Do