Enzymology – Part 1

Presented by Dr. Zahi Damuni

Professor of Biochemistry
[email protected]
TurningPoint/PointSolutions ID: damuni

Reading: Lippincott Reviews in Biochemistry, 8th Ed., Chapter 5

1
 Enzymology
Learning Objectives – Part 1
1. Explain in general terms how an enzyme speeds up a reaction.
2. Explain how temperature and pH affect the function of an enzyme.
3. Given two reactions with different enzymes and their associated kcat,
be able to identify which enzyme is “working” faster.
4. Describe the four general mechanisms of catalysis and identify them
from a reaction diagram.
5. Given a specific biochemical reaction, identify whether the enzyme is
a (an) oxidoreductase, transferase, hydrolase, lyase, isomerase, or
ligase.
6. Describe what cofactors are and how they can contribute to enzymatic
reactions.

2
 Enzymology Part 1 – Coverage
• What are enzymes?
• Basic Properties of Enzymes
• Catalytic Mechanisms of Enzymes
– Chymotrypsin, as an example
• Induced Fit versus lock and key
• Types of Enzymes
• Types of Cofactors

• Part 2: Enzyme Kinetics.

3
 Example
• A 58-year-old man visits his family physician for his annual checkup. The physician
orders routine blood tests, including lipids. It is found that the patients LDL-
cholesterol levels are high and this observation reflects a general increase in LDL-
cholesterol over time in this patient.
• The physician prescribes a statin drug. With follow-up tests, the statin was
successful in lowering LDL-cholesterol. This will decrease the risk of heart attack in
this patient.
• What do statins do?
• Why do they lower LDL-cholesterol?
• Where, exactly, do they act?
– Statins act on a specific enzyme to inhibit it.

~28% of adults over the age of 40 take statins (2012).

The percent of the population taking statins increases with age: 48% of adults over
75 take statins.

Statins act as enzyme inhibitors. They decrease the activity on an enzyme involved
in cholesterol synthesis.

Reference: https://www.cdc.gov/nchs/products/databriefs/db177.htm#1213132

4
 Examples of Enzyme-Catalysis
Creatine Kinase
Creatine Creatine-O-PO3 ADP
Creatine ATP PO3-
Kinase PO3-
PO3-
+ + + +

Example of an enzyme:
Creatine Kinase: The function of creatine kinase is to store high energy phosphate
bonds in creatine for fast conversion to ATP, when there is a high demand for ATP.
An easy example is a sprinter, who will very quickly go from resting muscle function
to very intense exercise. Creatine phosphate, which can be present in
concentrations as high as 20-35 mM (compared to 2-5 mM ATP), can replenish the
ATP from the creatine phosphate stores. This requires the enzyme, creatine kinase,
which is fast, and reversible.

The stored creatine phosphate helps maintain the ATP concentrations a few
seconds longer than otherwise. After that, the sprinter must rely on anaerobic
glycolysis to maintain ATP.

Key points:
Enzymes bind one or more substrates
Enzymes catalyze reactions on the substrates
Creatine Kinase creates creatine-phosphate to store high-energy bonds for fast use
in muscle.
The reaction can be rapidly reversed to regenerate ATP when it’s depleted.
This is an example of a transferase.

5
 Examples of Enzyme-Catalysis
Carbonic Anhydrase
10-2 sec-1
• Carbonic anhydrase facilitates
the dissolution of CO2 in red CO2 + H2O H2CO3 HCO3- + H+
blood cells and increases the
carrying capacity. E
• It is a metalloenzyme. 105 sec-1
CO2 + H2O H2CO3 HCO3- + H+

E E E
Overall rate enhancement = 107

Enzyme Example:
Carbonic anhydrase: This enzyme catalyzes the addition of water to CO2. It is an
example of a metallo-enzyme because it utilizes a zinc ion as part of its catalytic
mechanism.
The rate of dissolution of CO2 in and out of water can be slow. This is
seen when you open a soda. It doesn’t simply explode (unless you’ve shaken it),
but instead, the CO2 comes out of solution slowly, keeping the drink fizzy for some
time. Likewise, dissolving CO2 in water is slow, because it needs to be hydrated
(going from a gas to carbonic acid)
This is a problem physiologically, because CO2 has limited solubility
as a gas and needs to be hydrated to be carried efficiently (i.e. at higher
concentrations) in the blood stream. So, the slow addition and removal of water
limits CO2 carrying capacity without the presence of a catalyst to speed up
hydration. The enzyme is also critical for bicarbonate reabsorption in the kidney and
maintenance of pH.
The speed of the reaction can be compared here. The value is
referred to as the kcat, the catalytic rate constant. The rate in the absence of enzyme
is slow, 10-2 sec-1. This becomes 105 sec-1 in the presence of the enzyme, a 10
million-fold rate enhancement.

6
 Examples of Enzymes
Turnover rate: kcat
• Turnover rate: Approximate Turnover Numbers of Some Enzymes
– Fastest speed of an
enzyme Enzyme Turnover Number, kcat (s−1)
Carbonic anhydrase 600,000
– kcat determines the Vmax
Catalase 80,000
– The rate varies Acetylcholinesterase 25,000
substantially among Triose phosphate isomerase 4,400
enzymes. α-Amylase 300
– Reflects the catalytic Lactate dehydrogenase
200
power of the enzyme (muscle)
Chymotrypsin 100
– (Compare to the un-
Aldolase 11
catalyzed rate).
Lysozyme 0.5
Fructose 2,6-bisphosphatase 0.1
7

Enzymatic Rate Varies Greatly Among Enzymes

Turnover rate = kcat = k2
Enzymes can accelerate a chemical reaction enormously. Many reactions that
proceed within minutes in the presence of an enzyme would require thousands of
years to reach their equilibrium in the absence of a catalyst. The turnover number
describes the catalytic power of the enzyme. It is defined as the maximal number of
substrate molecules converted to product by one enzyme molecule per second.

The table lists the turnover numbers of some enzymes. (do not memorize these
numbers, they are just for example)

7
 General Properties of Enzymes
Enzymes remain unchanged after reaction.
• Enzymes bind their
substrate(s) Glucose + ATP Glucose-O-PO3- + ADP
(Glucose 6-phosphate)
• They act on the
substrates
• They release products
• They are unchanged at
the end of the
reaction
• (definition of catalyst).
S1 + S2 + E → ES1S2 → ES1S2‡ → EP1P2 → E + P1 + P2

An example from glycolysis: Hexokinase

This is an enzyme that illustrates many relevant enzymatic properties and we will
return to it as an example many times.

Hexokinase catalyzes the transfer of a phosphate from ATP to glucose to generate

glucose-6-phosphate

The key point of this slide is that the catalyst, hexokinase, is unchanged during the
net cycle of the reaction. It facilitates the transfer and emerges unscathed at the
end.
This does not mean the enzyme cannot be changed during the reaction. In many
cases, enzymes form covalent intermediates with their substrates or are modified in
some other way during the reaction. However, as a catalyst, these changes are
transient, and the enzyme is returned to its original state to facilitate another
reaction.

8
 General Properties of Enzymes
Temperature and pH Optima
• Proteins, including enzymes, typically have a
temperature optimum.
• Above and below this optimum, they are less stable,
will degrade faster, and have less activity.
• The optimum for many human enzymes is near 37
℃.
• Enzymes are typically pH sensitive
• Enzymes work optimally at a relevant pH:
– Low pH for pepsin to act in the stomach
– Moderately low pH for trypsin, a digestive
enzyme
– Alkaline phosphatase – very stable enzyme can
survive extremes
9

Temperature optima:
Proteins do not have a particular, obligatory temperature optimum. Some life forms
exist in sub-freezing temperatures; others exist near submarine volcanic vents at
temperatures near boiling. Yet, they all survive because they make proteins that
function in those environments.

However, evolutionarily, it is in an organism’s best interest to make efficient use of

proteins, so for humans, most proteins work most efficiently near 37 oC. Many of our
proteins will denature once you reach higher temperatures of more than 50 oC.

pH optima:
Similar to the idea of a temperature optimum, evolution has optimized pH
dependence of enzymes to be in the range of their environment.

Stomach enzymes have a low pH optimum because of the low pH environment.

Trypsin is a secreted enzyme, that functions at higher pH than that of stomach
acid.
Alkaline phosphatase, a general phosphatase, works best at high pH.

Most cytosolic enzymes will have pH optima near neutrality.

9
 Enzymology Part 1 – Coverage
• What are enzymes?
• Basic Properties of Enzymes
• Catalytic Mechanisms of Enzymes
– Chymotrypsin, as an example
• Induced Fit versus lock and key
• Types of Enzymes
• Types of Cofactors

• Part 2: Enzyme Kinetics.

10

10
 Mechanisms of Catalysis
Transition State Theory
• Transition State Theory
– A→ B
– DG reaction reflects overall reaction
equilibrium (independent of enzyme)
– Height of barrier reflects energy need
to achieve the Transition State energy
Ea or, more commonly, DG‡.
– Ea, or DG‡, is lower for the catalyzed
reaction.
– Lower DG‡ ➔ faster reaction.

11

Basic Thermodynamics of Reactions

Reminders from basic chemistry: The difference in energy between
the substrate and the product is the standard state free energy of the reaction: DGo.
In biochemistry, standard conditions of pH 7, and ignoring the water concentration,
and T = 25 oC. This is DGo'.
The actual DG of a particular reaction also depends on the
concentrations of the reactants, but in energy diagrams like this, we just focus on
the standard state free energy, DGo'.

Recall that DG is negative for a spontaneous forward direction, is positive for a

spontaneous reverse reaction, and is 0 at equilibrium.

This graph introduces another parameter, the transition state energy - DGact.
1. This is the height of the barrier to go from substrate to product. The barrier would
be higher in the reverse direction for this.
2. The height of the barrier corresponds the energy required to deform the substrate
into the transition state. The transition state is considered to be transient structure
where the compound is equally like to go back to substrate or go toward the
product.
3. The height of the barrier represents the energy needed to get to that structure.
4. The higher the barrier, the more energy is required. In general, the energy has to
come from Brownian motion in the environment, so the higher the barrier, the less
likely you are to get that much energy. Therefore, because the odds are lower of

11
getting that energy, the rate of going to product is smaller.
5. So, the higher the barrier, the slower the reaction. This is an exponential
relationship, so a small change in energy can still affect the rate a lot.

Enzymes are catalysts: Catalysts speed up the reaction.

So, effectively, catalysts lower the barrier (smaller barrier, faster rate).
That’s what the diagram shows. The catalyzed reaction has a smaller barrier.
This is one of the things enzymes do as catalysts, they reduce this barrier.
- How they reduce the barrier differs depending on the reaction.
- One of the means is that they actually stabilize the transition state itself,

Also remember, because catalysts are unchanged during the reaction, they cannot
put energy into the reaction. So, the basic energy difference between substrate and
product, the DG, remains unchanged. The enzyme can change the rate of the
reaction, but not how favorable it is.

11
 Mechanisms of Catalysis
General Mechanisms
1. Specific substrate interactions (approximation): Hexokinase Example
• binding reduces entropy
• proximity of substrates (substrate anchoring) increase in effective
concentration.
2. Binding transition state optimally: Chymotrypsin Example
• substrate deformation (induced fit of substrate)
• reduction of Transient State energy by strain or distortion
3. Providing covalent chemistry: Chymotrypsin Example
4. Providing Acid-base chemistry: Chymotrypsin Example

12

Catalytic Mechanisms
The general mechanisms of Catalysis are typically put in to 4 categories.
The categories are:
1. Specific substrate interactions – that is, only the substrate binds effectively. Other
compounds do not bind as well, even if similar in structure.
- Substrate specificity is critical for catalysts so as not to generate mis-reactions on
the wrong compounds.
- It is critical to provide a locally higher concentration of the substrates.
- It is critical to put the substrate in apposition, or near, the critical reactive residues,
either on the enzyme or on another substrate (or both).

One can think about how effective this is and quantify it. Consider a bimolecular
reaction – for example hexokinase, where both ATP and glucose have to be in the
binding site. In the hexokinase case, typically, glucose and ATP may be present in
~5 mM concentrations. Once bound to the enzyme, they are effectively present in
molar (M) concentration near each other.

- This apparent concentration boost, of both substrates of more than 100-fold can
give a very large rate increase (in this case > 104 sometimes even much more).
- One gets even more rate acceleration because they are oriented correctly for the
chemistry one bound.
- This concept has been tested chemically by comparing similar bimolecular versus
intramolecular reactions and similar rate changes are observed.

12
2. Binding the transition state optimally
- Refer to the transition state diagram shown earlier where the enzyme reduces the
barrier to the reaction. (Slide 7)
- This second catalytic mechanism is just the manifestation of this diagram.
- If the enzyme binds the transition state well, even better than substrate, what will
happen is that substrate will bind, and then deform a little, because of the binding
interactions. This deformation will bend the substrate into something that looks more
like the transition state. This effectively lowers the barrier to achieve the transition
state and is caused by the binding interactions with the enzyme.
- So, properly positioned residues can stabilize the transition state and lower the
barrier to reaction.

3. Providing covalent chemistry:

For many reactions, the spontaneous rate in water would be slow because the
reagents needed to make the reaction go are not present, or only present in very low
concentration. By reacting with the enzyme, the enzyme can provide the reactive
functional groups and position them properly. Covalent intermediates also serve to
hold a substrate in place, sometimes so a second substrate can enter and complete
the reaction. Again, this is equivalent to providing a locally high concentration (like in
the first mechanism).

4. Providing acid/base chemistry:

Many reactions are facilitated by acids or bases, but most biological reactions occur
under cellular conditions near neutral pH where both acid and base concentrations
are quite low. By making side chain residues basic or acidic and placing them in the
substrate binding site in a proper position, the chemistry can be facilitated and
enhanced by many orders of magnitude.

The categories are not completely independent factors, but each are conceptually
distinct, which helps us think about them.

12
 Mechanisms of Catalysis
Specificity and Induced Fit – Hexokinase
• Enzymes are highly specific
for their substrates
• Even very similar
compounds are typically
excluded from the
reaction:
– Water: looks like
glucose hydroxyl
– Ribose: similar sugar
molecule HO
OH
O
• This is critical for function
in Biology
OH OH
13

Substrate Specificity
Substrate specificity for enzymatic reactions, and for general binding
recognition, is the idea that protein only acts on a particular molecule. For example,
most kinases will use ATP to transfer a phosphate to another molecule. Most cannot
use GTP, even though it is a closely related molecule. We therefore say that such a
kinase has specificity for ATP over GTP. The specificity of enzymes for particular
substrates is what permits metabolism to work efficiently and in a highly directed
manner, where you can enter a pathway (such as glycolysis), go through many
intervening enzymatic steps, wind up with a particular product at the end, produced
with little to no wastage on irrelevant by products.
Notably, in some enzymes, there is some overlap in specificity. This is
some cases purposeful, such as a cytochrome p450 hydroxylating many kinds of
substrates prior to excretion. In other cases, the enzyme can process several
similar kinds of substrates (e.g. alcohol dehydrogenase works on both methanol and
ethanol).
The so-called lock-and-key binding mechanism is where the enzyme
is a “best” complimentary fit for the substrate, as proposed my Emil Fisher. That is,
various functional groups in the enzyme are perfectly juxtaposed to interact
favorably with functional groups on the substrate.

Specificity is now thought to be achieved in enzyme catalysis through additional

mechanisms: The induced fit hypothesis.
- Specificity is achieved not only by complementary interactions of various functional

13
groups, but also through subsequent changes in protein structure.
- This is called the induced fit hypothesis. See the next slide for a movie illustrating
this.

In Hexokinase, Daniel Koshland considered its mechanism and wondered how it was
that it could efficiently catalyze the transfer of phosphate to a glucose hydroxyl group,
but wouldn’t do the same for a water. After all, he thought, water and glucose
hydroxyl are chemically pretty similar, and water is present at 55 M, a concentration
roughly 10,000 times that of glucose. Obviously, if the enzyme didn’t have this
specificity, ATP would just be hydrolyzed by reacting with water and the energy
wasted. So, some mechanism built into the protein existed to prevent this.
The mechanism he proposed is called the induced fit hypothesis. The
rationale is that when the correct ligand binds, the binding interactions draw the
enzyme into a new conformation that properly aligns the catalytic residues so they
can carry out the chemistry. The ligand, glucose, changes the structure of the
enzyme from an inactive form to an active form.

Approximation
This concept goes along with specificity. The ideal is that in most
biological situations, the substrate concentrations are rather low, especially when we
compare them to what we typically do in chemistry labs. Because the concentrations
are low, the chance that two molecules, such as ATP and Glucose, encounter each
other and react, are quite modest. The higher the concentration of either substrate,
the higher the odds of an encounter that leads to reaction. With specific binding, the
enzyme effectively does this for you. It can bind each of these molecules, and ina
way that aligns them for reaction. This can be thought of as effectively increasing the
local concentration of reactants (within the enzyme). This can provide a large amount
of the catalytic increase. Consider that ATP and glucose may both be present around
5 mM. In an enzyme and aligned properly, they are likely near effective 1 molar
concentrations (or perhaps higher). This is each a 200 fold increase. Multiplied
together, this effect alone may produce as much as a 40,000-fold increase in reaction
rate. Thus, approximation through specific binding can contribute a large amount of
the catalytic effect of enzymes.

13
 Mechanisms of Catalysis
Induced Fit – Hexokinase movie
• Video of Glucose binding to
the catalytic domain of
Hexokinase:
• Binding of glucose induces
a conformational change:
– Activates the active site
– Facilitates binding of
ATP

Pedersen 2021 September 14

Induced Fit Binding

Hexokinase binds glucose in an induced fit manner. This means that when glucose,
the substrate binds, it interacts with the protein in a way that changes the proteins
shape. The hexokinase molecule is dynamically flexible and will adopt a new shape
to enhance the binding interactions with glucose.
The change in shape can have several effects: It can move reactive
residues into place, it can stabilize the transition state of the substrate (the substrate
can also change its shape a bit), and it can increase the binding of co-substrates
(ATP in this case).

Lock and Key Binding

Originally proposed by Fisher as the means by which enzymes recognized their
substrates. Lock and key binding refers to binding that has good complementarity
without a change in shape induced by the binding.

Both mechanisms occur, but induced fit binding is important for most enzyme
mechanisms.

14
 Mechanism of Chymotrypsin
• Chymotrypsin:
The overall reaction is:
– Enzyme localized
in the small
intestines O O
– Cleaves peptide
after aromatic …-NH-C1HR-C-NH-C2HR-C-…
residues.
H2O
• Example of both: O O
– Lock and key
…-NH-C1HR-COH NH2-C2HR-C-…
binding
– Induced fit binding

15

There are numerous examples of covalent bond formation and acid/base catalysis
during enzymatic reactions.
A classical example which combines both is proteolysis by a serine protease. For
example, trypsin, chymotrypsin, pepsin, subtilisin, etc.
These enzymes show features of lock and key binding for specificity,
Of induced fit binding for transition state stabilization
Of Covalent chemistry
And both acid and base catalyzed chemistry.

15
 Mechanism of Chymotrypsin
• Steps:
– Initial Binding
– Base-catalyzed nucleophilic attack
– Tetrahedral intermediate
(transition state 1)
– Acid-catalyzed release of C-
terminus
– Formation of Acyl-enzyme
– Water entry
– Base catalysis forms second
Tetrahedral intermediate
– Acid-catalysis releases N-terminal
fragment.
16

Note – You do not have to be able to reproduce these reactions.

You do have to be aware of the types of catalytic mechanisms and this is an
illustration of several of them.

16
 Mechanism of Chymotrypsin

• Lock and Key binding in serine

proteases:
– Chymotrypsin – for aromatic or
hydrophobic residues: Trp, Tyr,
Phe, and Leu.
– Trypsin – for Lys and Arg (cationic).
– Elastase – small Hydrophobic
residues: Gly, Val, Ala.
• Induced fit binding may occur within
the active site.

17

• The binding site holds the molecule to be changed in such a way that the catalytic
site is in the correct position to react with the molecule and change the molecule
into a product.
• Trypsin has a (-) charged aa side chain that forms an ionic bond with a (+)
charged aa in the substrate.
• Chymotrypsin has a small side chain that allows large hydrophobic residues
to bind.
• Elastase has hydrophobic side chains that interact with small
hydrophobic side chains in the substrate.
• These interactions position the active site Ser in the right place to allow
the –OH on its side chain to cleave bonds in the substrate.
• Note that the “serine protease” cleaves the next peptide bond on the C-terminal
side of the amino acid that binds to the “binding pocket” of the active site of the
enzyme.
Transition state stabilization by the oxyanion hole
Also important in this slide in the top right figure is the Oxyanion hole.
These are backbone or side chains that interact with the carbonyl of the peptide
bond.
These interactions help stabilize the tetrahedral transition state. This residues
therefore help distort and conform the substrate to the transition state and stabilize
it. This lowers the Transition state barrier and increases the rate of reaction.

17
An oxyanion hole is a pocket in the active site of an enzyme that stabilizes
transition state negative charge on a deprotonated oxygen or alkoxide. The
pocket typically consists of backbone amides or positively charged residues. The
oxyanion hole of serine proteases is formed by the backbone N atoms of the
catalytic Ser-195 and Gly-193 and engages the backbone O atom of the P1
residue of substrate in an important H-bonding interaction.

17
 Enzymology Part 1 – Coverage
• What are enzymes?
• Basic Properties of Enzymes
• Catalytic Mechanisms of Enzymes
– Chymotrypsin, as an example
• Induced Fit versus lock and key
• Types of Enzymes
• Types of Cofactors

• Part 2: Enzyme Kinetics.

18

18
 Classification of Enzymes
• Oxido-reductases
– HMG-CoA reductase, the rate-limiting step in cholesterol
synthesis
• Transferases
– Protein Kinase A (PKA), Protein kinase G (PKG)
• Hydrolases
– Amylase, Trypsin, Lipase, RNAase, DNAase, Lactase,
• Lyases
– Histidine decarboxylase
• Isomerases
– Phosphogluco-isomerase
• Ligases
– DNA, RNA ligases, DNA and RNA polymerases, Pyruvate
carboxylase

19

Note – Knowing these classes and understanding how enzymes fit into them is
required USMLE knowledge.

19
 Oxido-reductases
• Oxido-reductases
– Example: Alcohol
Dehydrogenase
– Detoxifies ethanol.
– Uses NAD+ as a cofactor
– An example of an
oxidation
– Ultimate product will be
acetate which is
metabolized.

Human ADH5 20

Oxido-reductases –
Facilitate redox chemistry.
Often involve NAD or FAD
NAD requiring enzymes are called dehydrogenases (if you see this
name, you know its an NAD requiring oxido-reductase).

The example at the right is lactate dehydrogenase, an enzyme that converts

pyruvate to lactate during anaerobic metabolism in muscle, and reverses the
reaction in the liver

20
 Transferases
• Transferases
– Example: Glycerol Kinase
– Primes Glycerol for
conversion to glucose in the
liver during gluconeogenesis

21

Transferases move a group from one molecule to another.

A common example is kinases: They transfer a phosphate group from one molecule
(ATP typically), to another. Shown is the liver enzyme, glycerol kinase. Another
example is protein kinase A, which will catalyze the transfer of phosphate from ATP,
to regulated proteins.

Another example involved in protein catabolism is transaminases, which move an

amino group from one molecule to another.
The example at the right, serine hydroxymethyl transferase transfers a hydroxyl
methyl from serine to THF, tetrahydrofolate, producing glycine

21
 Hydrolases

• Hydrolases
– Example: Protease
– Includes: Esterases, proteases, lipases, sugar hydrolases, and
ether hydrolases.

• Key: addition of water.

22

Hydrolases
These use water addition to break a chemical bond.
Proteins in this class include those that break peptide and ester bonds. For
example, proteases, esterases, and lipases.

Typically, one molecule is cleaved into two new products.

Also included are nucleases, phosphodiesterases, helicases, DNA glycosylases.

There are many others.

Do not confuse with hydroxylases, which add a hydroxyl group.

22
 Lyases
• Lyases
– General examples:
• Dehydratases
• Decarboxylases
Dehydratase

Pyruvate Decarboxylase

23

Lyases:
Catalyze cleavage of C-C bonds, C-O bonds, C-N bonds, C-S bonds, and C-halide
bonds. Also, P-O bonds.
Decarboxylases are one example. Dehydratases are another (remove water; breaks
C-O bond).

Pyruvate decarboxylase is part of the pyruvate dehydrogenase complex or acts

alone to convert pyruvate to acetaldehyde and further conversion to ethanol during
fermentation in yeast or bacteria.

23
 Isomerases

• Isomerases
– Phosphoglucomutase

PhosphoGlucomutase
(Wikipedia)
24

Isomerases
As the name implies, isomerases catalyze isomerization reactions, often among
stereoisomers to racemize them.

The example of phosphoglucomutase, shows isomerization of glucose-6-phosphate

to glucose-1-phosphate. This is a readily reversible enzyme that functions near
equilibrium.
Isomerases typically have one substrate and one product.

The example on the right is methylmalonyl CoA mutase which converts this
intermediate to the TCA cycle intermediate, Succinyl CoA.

24
 Ligases
• Ligases
– Example: Glutamine Synthetase
– Catalyzes transfer of ammonium to
Glutamate
– Glutamine is a major nitrogen
carrier in the blood

Pedersen 2021 September 25

Ligases
Catalyze the formation of bonds: C-O, C-S, C-N, C-C and others.

The example shown is glutamine synthetase which adds ammonium to the amino
acid glutamate.
This is an important reaction for the movement of nitrogen within the body.

The example on the right is from the initial step in gluconeogenesis whereby
Pyruvate is converted to oxaloacetate, at the expense of an ATP.
This reaction can also be used to replenish TCA cycle intermediates in case their
concentration drops. Those reactions that replenish the TCA cycle are called
anaplerotic reactions.

25
 Enzymology Part 1 – Coverage
• What are enzymes?
• Basic Properties of Enzymes
• Catalytic Mechanisms of Enzymes
– Chymotrypsin, as an example
• Induced Fit versus lock and key
• Types of Enzymes
• Types of Cofactors

• Part 2: Enzyme Kinetics.

Pedersen 2021 September 26

26
 Cofactors
Example: Coenzyme A
• Coenzyme A – combination of ADP, pantothenic acid, and cysteamine
• Acts as a carrier for high-energy acyl bonds
• Pantothenic acid is vitamin B5 (rarely deficient).
Cysteamine Pantothenic acid

3’-phosphoADP 27

The role of cofactors

In general: Cofactors help facilitate reactions where unique reactivity is required, or
reactivity not readily supplied by the canonical set of 20 amino acids.

Cofactors, coenzymes, and prosthetic groups all have this purpose but are defined
slightly differently:
1. Cofactor is the most general inclusive term and refers non-protein compounds,
metals, or ions needed for enzymatic activity. Sometimes ‘cofactor’ is used to
refer only to inorganic ions to distinguish them from coenzymes
2. Coenzymes are organic molecules that serve as cofactors. These are often
derived from vitamins.
3. Prosthetic groups are coenzymes that remain tightly bound to an enzyme or
protein . An example is heme binding to hemoglobin.
4. Cosubstrates are coenzymes that bind transiently and may be released from the
protein. NAD+ is an example.

Coenzyme A
Used as an acyl carrier and is involved in many catabolic and anabolic reactions.
The key reactive part is the sulfhydryl at the end.

27
 Cofactors
Examples: NAD and FAD
• NAD+ - Nicotine-Adenine dinucleotide.
• NADP+ - phosphate added.
• FAD – Flavine-adenine dinucleotide
• Carry electrons and Facilitate redox reactions.

28

Vitamin-based cofactors.
Examples of the two most common redox carriers: NAD and FAD
These are both nucleotides, using a base/ribose/phosphate structure.
The also have a ‘dinucleotide’ structure where two sugars are joined through a
diphosphate ester linkage. In the case of FAD, the sugar is ribitol rather than ribose.
Both the bases here – Nicotinamide and Flavine, are derived from vitamins: niacin
and riboflavin.

A key functional difference between the two is that:

NAD/NADH is typically used as a soluble carrier of redox equivalents and acts
more as a substrate than cofactor in many reactions.
FAD/FADH2 is typically constitutively bound as a cofactor to proteins when used
as a redox carrier.
NAD has a more negative redox potential than FAD and provides more energy
per electron carried for ATP production.

28
 Coenzyme Present as Functions in Vitamin*
Enzymology Adenosine triphosphate (ATP) Co-substrate Energy metabolism —
Guanosine triphosphate
Enzymatic Cofactors (GTP)
Co-substrate Energy-metabolism —
Activation of
Uridine triphosphate (UTP) Co-substrate —
monosaccharides
• Various Cofactors Cytidine triphosphate (CTP) Co-substrate
Phospholipid
—
synthesis
– Many are vitamins Nicotinamide adenine
– All carry out special dinucleotide (NAD) and
(NADP)
Co-substrate Hydrogen transfers Niacin

functions not easily Flavin adenine dinucleotide

Prosthetic group Hydrogen transfers Riboflavin
supplied by the (FAD) also (FMN)
Pantothenic
canonical 20 AA set. Coenzyme A Co-substrate Acylation reactions
acid
S-adenosyl methionine (SAM) Co-substrate Methylation Rxns —
Heme Prosthetic group Electron transfers —
Carboxylation
Biotin Prosthetic group Biotin
reactions
One-carbon
Tetrahydrofolate (THF) Co-substrate Folic acid
Summary of the transfers
Most Important Amino acid
Pyridoxal phosphate (PLP) Prosthetic group B6
Coenzymes metabolism
Thiamin pyrophosphate (TPP) Prosthetic group Carbonyl transfers Thiamin (B1)
Oxidative 29
Lipoic acid Prosthetic group —
decarboxylations

As cofactors are introduced in the discussion of specific enzymes in various

pathways in later lectures, you will have to know how and where most of these are
important. So it won’t hurt to familiarize yourself with these.

29
Vitamins can act as coenzymes that participate in catalysis by providing functional groups. Therefore,
vitamin deficiencies reflect the loss of specific enzyme activities that depend on those coenzymes.
Coenzymes are best described by which one of the following?

A. In humans, they are always

synthesized from vitamins.
B. They are proteins.
C. They participate in only one reaction,
like enzymes.
D. They are complex, nonprotein organic
molecules.
E. They are all carbohydrates.

30

30
 Enzymology Summary
• Enzymes are catalysts
• Enzymes catalyze through binding, stabilizing the transition state,
facilitating covalent chemistry, and also facilitating acid, base chemistry.
• Enzymes are classified based on the type of reaction they catalyze
• Cofactors and coenzymes help facilitate reactions.

31

31