Enzyme Reaction

Mechanisms
•Lehninger A.L., principles of
biochemistry, 5th edition
Lecture outcomes:

1. Explain models of enzyme substrate interaction

2. Describe energy changes during reaction

3. Outline the four principal catalytic mechanisms and how they

can be combined by enzymes to facilitate chemical reactions.

What is an enzyme reaction mechanism:

The particular chemistry that an enzyme uses to catalyze a

reaction.

How the substrate interacts with the enzyme to produce

catalysis.

The nature of the amino acids from the enzyme that are
involved in catalysis.

The nature of the “transition state” of enzymatic

catalysis.
 MECHANISM OF
ACTION
• Enzymatic catalysis of biological reactions is
essential to living systems
• Enzymes hasten chemical reactions or they
affect rate of a reaction without affecting the
equilibria
E +S ES → P + E
• Where E = enzyme, S= substrate, ES=
enzyme substrate complex, P= product
• NB: the substrate attaches to enzyme active
site and forms ES complex before
transformation to product
 Models of Enzyme-Substrate
• interaction
Two models have been suggested for
the interaction of enzymes with their
substrates
1) Lock and Key model:
 It is assumed that the substrate and the
active site have structural similarity as a
key fits its padlock.
2) Induced fit model:
 Approaching of the substrate to an
enzyme active site brings about a
conformational change on the active site.
This model better explains properties of
 Induced fit model

Binding of the substrate A—B induces conformational

changes in the enzyme that align catalytic residues
which participate in catalysis and strain the bond
between A and B, facilitating its cleavage.
 Mechanism of
Energy changesaction…
during the reaction
• Enzymes decrease the energy of activation
(Ea) for a reaction and hence speed up the
rate of reaction
• Enzymes do not affect the thermodynamics (DG) of
the reaction (net free energy change for the
reaction or equilibrium concentrations of the
substrates and products).
• The transition state is at the apex (the top) of the
energy diagram between reactants and products.
 Mechanism of
action…
• The difference in the average free energy of
the reactants and the average free energy of
the transition state is the activation energy
barrier (free energy of activation; Ea).
• The lower the EA, the more molecules will have
sufficient energy to surmount the energy
barrier, the fastest the rate of reaction
What is the source of the energy for the dramatic lowering of the
activation energies for specific reactions?

1.Binding energy, ΔGB: noncovalent interactions

between enzyme and substrate binding energy is a
major source of free energy used by enzymes to lower
the activation energies of reactions.
2.Covalent interactions between enzyme and
substrate
Types of Catalytic Mechanisms
1.Acid-base catalysis;Specific" acid-base catalysis
involves H+ or OH- that diffuses into the
catalytic center. "General" acid- base catalysis
involves when acids and bases transfer H+ in the
transition state
2.Covalent catalysis; formation of a covalent bond
between the enzyme and one or more substrates
(lowers the activation energy)
3.Metal ion catalysis; Ionic interactions between an
enzyme- bound metal and a substrate can help
orient the substrate for reaction or stabilize
charged reaction transition states.
4.Proximity and orientation effects; When an
We will look at mechanisms of 4 enzymes:
1. Chymotrypsin ; illustrates the principle of transition-state
stabilization, general acid-base catalysis and covalent
catalysis.

2. Hexokinase – classic example of induced fit model

3. Enolase – classic example of metal-ion mediated catalysis

4. Lysozyme --classic example of SN2 chemistry and stereo-

specific hydrolysis and acid base catalysis
 Enzyme Mechanisms – Serine
Proteases
• Proteolytic enzymes help degrade proteins and
recycle amino acids in living systems.
• The serine proteases are an important sub-
group of this class of enzymes. The alcoholic
functional group of serine at the active sites of
these proteases serves as a strong nucleophile,
attacking the carbonyl carbon in peptide
bonds.

1
3
Enzyme Mechanisms – Serine
Proteases
Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin

• All involve a serine in catalysis - thus the name

• Ser is part of a "catalytic triad" of Ser, His, Asp
• Serine proteases are homologous, but locations of
the three crucial residues differ somewhat
• Enzymologists agree, however, to number them
always as His-57, Asp-102, Ser-195
• Burst kinetics yield a hint of how they work!
Enzyme Mechanisms – Serine
Proteases
A mixture of covalent and general acid-base catalysis
• Asp-102 functions only to orient His-57
• His-57 acts as a general acid and base
• Ser-195 forms a covalent bond with peptide to be
cleaved
• Covalent bond formation turns a trigonal C into a
tetrahedral C
• The tetrahedral oxyanion intermediate is stabilized by
N--H of Gly-193 and Ser-195
1
6
 Enzyme Mechanisms – Serine Proteases

• Reagents such as
diisopropylphosphofluoridate (DIPF) that
react with serine can “poison” these
enzymes, rendering them inactive:

1
7
 Chymotrypsinogen… Zymogen

• Chymotrypsinogen is composed of a single

polypeptide chain with 245 amino acid residues
and 5 intra chain disulfide bridges.
• Chymotrypsin is one of the best known serine
proteases.It catalyzes the hydrolysis of peptide
bonds following amino acids with large, bulky non
polar groups (e.g., phenylalanine
• Mechanism involves acylation and deacylation
of a ser residue.
Chymotrypsin:
• Serine protease, specific for peptide bonds adjacent to
aromatic amino acids
• Enhances the rate of peptide bond hydrolysis by
more than 109 (a billion)
• Uses special kind of hydrolysis that does not use the
direct attack of water on the peptide bond
• The hydrolysis uses a transient covalent acyl-enzyme
intermediate
Chymotrypsin: mechanism

Chymotrypsin can be tricked into hydrolyzing synthetic

substrates that release a highly colored substrate such as p-
nitrophenol. This facilitates its study in the laboratory.

Reaction mechanism is split into two phases:

Acylation phase: peptide bond is cleaved and an ester linkage

is formed between the peptide carbonyl and the enzyme

De-acylation phase: ester linkage is hydrolyzed and the non-

acylated enzyme is regenerated, allowing another round of
catalysis.
 Enzyme Mechanisms –
Chymotrypsin
Reaction mechanism is split into two phases:

Acylation Step 1

De-acylation Step 2

Enz +

Can we see these differing rates when we examine the

kinetics of chymotrypsin? Yes
 Enzyme Mechanisms – Chymotrypsin

• Ser-195 attacks substrates, forming an ester linkage to the

substrate as the first step in the reaction mechanism. This
leaves part of the substrate covalently bonded to the enzyme.
• Water subsequently enters, deacylating the enzyme by
hydrolyzing the ester bond.

2
2
 Enzyme Mechanisms –
Chymotrypsin
Kinetics of the enzyme suggest TWO rates of reaction:

Two different rates of reaction change the shape of the

kinetic profile
 Enzyme Mechanisms –
Chymotrypsin
• The first step of this reaction is
FAST. The rate-limiting step is
hydrolysis of the ester bond to
free the enzyme for the next
cycle.

• This is shown by rapid mixing

experiments that allow rate
determinations at the
millisecond time scale.
“Burst Phase” kinetics at time
zero, change to a slower rate
after all enzymes are acetylated,
waiting for water to release them
in the rate limiting step:
Hexokinase:
Adds phosphate group onto glucose to form glucose 6-phosphate:

Has two substrates H20 and glucose, but favors reaction with
glucose by 106

Enzyme can discriminate between glucose and H20 because of a

conformational change in the enzyme when GLUCOSE binds, but
not H20
Hexokinase:
Enzyme undergoes induced fit when glucose binds:

When the enzyme is put in the proper conformation, it is then

ACTIVE, and will then ADD PHOSPHATE to glucose.
Hexokinase:
Enzyme undergoes induced fit when glucose binds:
However, when Xylose (5 carbon sugar) binds, hexokinase also
adopts(choose) ACTIVE conformation

Carbon 6 of Glucose is missing from Xylose, so hexokinase will

phosphorylates WATER instead.
Enolase:
Catalyzes the dehydration of 2-phosphoglycerate to
phosphoenolpyruvate:

Good example of metal ion catalysis – the use of the positive

charge on metal ions to stabilize the high-energy intermediate
Enolase:
Reaction occurs in two steps:

Step 1: Proton extraction from phosphoglycerate by Lysine(345) of

Enolase enzyme.

Setp 2: Proton donation from Glutamate(211) of the Enolase

enzyme to the –OH leaving group.
Enolase:
Reaction occurs in two steps:

Proton Proton
Extraction Donation
NB: Metal ions (Mg2+) help stabilize the enolic intermediate
(two negative charges on the two oxygens).
Lysozyme: (bacteria LYSing enZYME).
• Is an enzyme that degrades bacterial cell walls.
• It Hydrolyzes β(1->4) glycosidic bond from N-acetylmuramic
(NAM) acid to N-acetylglucosamine (NAG) in cell wall
peptidoglycan.
• It also hydrolyzes chitin: β(1->4) NAG.
• Natural antibacterial agent found in human tears and in egg
whites.
• First enzyme to have 3D crystal structure solved in 1965
by X-ray crystallography
• Substrate is peptidoglycan, a carbohydrate found in
bacterial cell walls
• Cleaves the ( -1,4) glycosidic linkage between the
 Cont’d…
• The scientist believed that this enzyme might be an
excellent antibiotic for treating bacterial infections.
Lysozyme cleaves polysaccharides that give
structural integrity to bacterial cell walls.
• Cell wall polysaccharides are composed of two
kinds of glucose derivatives connected by
β(1→4) linkages:
NAG: N-
acetylglucoseamine
NAM: N-acetylmuratic
acid
 Lysozyme occurs widely as bactericidal
Lysozyme:
Six residues of the repeating disaccharide
bind to the active site of lysozyme:

Cleavage occurs between the D

and E sugars, numbers 4 & 5
Lysozyme: Glu35 acts as a general acid.
Asp52 stabilizes a carbonium ion intermediate.
Uses SN2 chemistry to catalyze lysis of (1,4) link:
Lysozyme:
Uses SN2 chemistry to catalyze lyses of (1,4) link:
 Summary
• Chymotrypsin is a serine protease with a well understood
mechanism, featuring general acid base catalysis, covalent
catalysis, and transition-state stabilization.
• Hexokinase provides an excellent example of induced fit as a
means of using substrate binding energy.
• The enolase reaction proceeds via metal ion catalysis.
• Lysozyme makes use of covalent catalysis and general acid
catalysis as it promotes two successive nucleophilic
displacement reactions.
 Factors Affecting
rate
 Physical and chemical factors affect
the enzyme activity
1) Substrate concentration: Rate of
enzyme catalyzed reaction increases
with increase in [S] until the
maximal velocity (Vmax) is reached.
NB: after Vmax is reached increasing the
[S] will not have any effect on
reaction rate due to saturation (active
sites are occupied by reactants
forming ES).
Figure: Representation of an enzyme in the presence of a
concentration of substrate that is below Km (A), at a
concentration equal to Km (B), and at a concentration well
above Km(C).
 Factors
affecting….
2) Temperature:
• Increase in T increases reaction
rate to reach maximum activity due
to increased number of molecules
having sufficient energy
– further increase lowers the rate
due to denaturation of the
enzyme

The T at which the enzyme is 100%

active is termed the optimum
temperature.
 Factors affecting
3)pH:
…
Same to T pH is required for
enzyme activity but further increase
in pH due to denaturation of enzyme
brings in sharp decline in enzyme
activity hence reaction rate.
NB: The pH at which an enzyme is
100% active is termed the
optimum pH.
– The optimum pH varies for different
enzymes.
Some are active at neutral pH some at
acidic pH and some at basic pH.
The pH-activity profiles of two
enzymes
 Enzyme
kinitics
• Harper’s Illustrated Biochemistry-McGraw-Hill
Education _ Medical (2018), 31st edition.
• Lehninger A.L., principles of biochemistry, 5th
edition 2013
 Lecture
1. outcomes
Define initial rate conditions and explain the advantage obtained from
measuring the velocity of an enzyme-catalyzed reaction under these
conditions.
2. Describe the importance of Km and Vmax, and their relations with the
substrate concentration
3. Describe the application of linear forms of the Michaelis-Menten equation to
estimate Km and Vmax.
4. Describe the effects of competitive, noncompetitive and uncompetitive
inhibitors on Vmax and Km
5. Contrast the effects of an increasing concentration of substrate on the kinetics
of simple competitive, noncompetitive and uncompetitive inhibition.
6. List and explain the different mechanisms that regulate enzyme activity.
7. Describe the clinical applications of enzymes
 Enzyme
Kinetics
• The field of Biochemistry that studies the
quantitative measurement of the rates of
enzyme-catalyzed reactions and the
systematic study of factors that affect
these rates.

• Kinetic analyses permit scientists to

reconstruct the number and order of the
individual steps by which enzymes
transform substrates into products.
 Enzyme
Kinetics…
• Enzymes lower the Energy of Activation
but do not affect the equilibrium.
• In presence of the enzyme (E) the
equilibrium constant for a reaction,
A + B + Enz →P + Q + Enz

Keq = [ P] [Q] [Enz]

= [P] [Q]
A. [B] [Enz] [A][B]

NB: The enzyme has no effect on the

Keq as it cancels out in the equilibrium
 Enzyme
Kinetics…
• Measuring rate is so difficult as
concentration of substrate [S] varies
during the course of reaction.
• We assume steady state i.e. a state where the
enzyme-substrate complex concentration [ES]
and other intermediates becomes a constant over
time
• We measure initial velocity by taking different
[S] each time.
• At one point increase in [S] will not have any effect on
the rate because of saturation
– All active sites on enzyme occupied by substrate.
This velocity is termed the maximum possible
velocity (Vmax) of that enzyme catalyzed reaction.
THE MICHAELIS-MENTEN
EQUATION
The steady-state assumption
k1 k2
E+ ES E+
S k-1 P

1. Rate of formation of ES d[ES] dt = k1 [E] [S]

complex
-d[ES] dt = (k-1 + k2)
2. Rate of destruction ES complex
k1 ([E]t- [ES]) [ES]
[S] = (k-1 + k2)
3. Steady State Equilibrium [ES]
k-1 + k2 = ([E]t- [ES])
4. Abbreviate kinetic constants k1 [S]
as Km Km [ES]
5. Solve for [ES = [E]t [S]
[ES ] ] Km + [S]
6. Substitute in v = k2 [ES] v = k2 [E]t [S]
above Km + [S]
7. Substitute Vmax for k2 v = Vmax [S]
[E]t Km +
[S]
50
Vmax = velocity where
all of the enzyme is
bound to substrate
(enzyme is saturated
with S)

Km = [S] @ ½
Vmax (units
moles/L=M) (1/2 of
enzyme bound to
S)
 Cont…

o The theoretical maximal velocity

• Vmax is a constant

• Vmax is the theoretical maximal rate of the

reaction
– but it is NEVER achieved in reality
• To reach Vmax would require that ALL
enzyme molecules are tightly bound
with substrate
 Cont
… constant"
The "kinetic activator
• Km is, an estimate of the
dissociation constant of E from S
for a given enzyme
• It is the concentration of the
substrate at which a given enzyme
yields one-half its max velocity
• Reflects the affinity of the
enzyme for its substrate.
• Small Km means tight binding;
high Km means weak binding
 Enzyme
kinetics
• Small Km value reflects a high affinity of the
enzyme for substrate and high Km reflects low
affinity.
• The rate of the reaction is directly proportional
to the enzyme concentration at all substrate
concentrations.

• Only initial reaction velocities (vo)are used

in the analysis of enzyme reactions

 Enzyme
• When [S]<<[Kkinetics
m] the reaction velocity is
proportional to the [S], the rate depends on [S]
and the order of the reaction is first order.

• When [S]>>[Km] vo = Vmax the reaction

velocity becomes independent of [S] and the
reaction is zero order.

• When [S] = Km the initial velocity is half

 The turnover number - measure of catalytic activity
• kcat, the turnover number, is the number of
substrate molecules converted to product per
enzyme molecule per unit of time, when E is
saturated with substrate.
• If the M-M model fits, k2 = kcat = Vmax/Et
• Values of kcat range from less than 1/sec to many
millions per sec
The catalytic efficiency - Name for kcat/Km
 An estimate of "how perfect" the enzyme is
 kcat/Km is an apparent second-order rate constant
 What does kcat/Km
mean?
• It measures how the
enzyme performs when S is
low
• kcat/Km describes an enzyme
preference for different
substrates = specificity
constant
• The upper limit for kcat/Km is the
diffusion limit - the rate at
which E and S diffuse together
(108 to 109 M-1s-1)
• Catalytic perfection when
kcat/Km
= diffusion rate
 Limitations of M-M
1. Some enzyme catalyzed rxns show more complex
behavior
E + S<->ES<->EZ<->EP<-> E + P
With M-M can look only at rate limiting step
2. Often more than one substrate
E+S1<->ES1+S2<->ES1S2<->EP1P2<-> EP2+P1<->
E+P2
Must optimize one substrate then calculate
kinetic parameters for the other
3. Assumes k-2 = 0

4. Assume steady state conditions

 Linear Plots of the Michaelis-Menten
Equation
Because of the difficulty of exactly determining Vmax from
a hyperbolic curve, the Michaelis-Menten equation was
transformed into linear plots
Lineweaver-Burk
Lineweaver-Burk Plots
(double reciprocal plots)

•Plot 1/[S] vs 1/Vo

• L-B equation
for straight line
•X-intercept = -1/Km
•Y-intercept = 1/Vmax
• Easier to
extrapolate values w/
straight line vs
hyperbolic curve
 V
max
0.2
5 B
B B [S] Vo
0. B 0. 0.07
2 5 5
0. 0.09
0.1 B
75 0.15
5
2 2
Vo

4 0.19
0.
B 6 6
1
B 8 0.21
0.0 10 0.21
K 4
5 Km ~ 0.23
1.3
m
0
mM
0 1 2 3 4 5 6 7 8 9
10 Vmax ~
[S] 0.25
 1
4 B
1
2 B 1/[S] 1/Vo
1 2.000 13.33
0 3
1.333 11.11
1/Vo

1
8
B 0.500 6.579
6
0.250 5.102
0.167 4.762
4 B
BB
0.125 4.673
B
0.100 4.348
2
-1/Km = -0.8
0 B Km = 1.23
-1 -0.5 0.5 1 1. 2 mM
0 1/[S] 5
1/Vmax =
4.0
 Enzyme
Inhibition
• Inhibitor – substance that binds to an enzyme
and interferes with its activity
• Can prevent formation of ES complex or
prevent ES breakdown to E + P.
• Irreversible or reversible inhibitors
• Irreversible inhibitor binds to enzyme through
covalent bonds (binds irreversibly)
• Reversible Inhibitors bind through non-
covalent interactions (disassociates from
enzyme)
• Why important?
 Reversible Inhibitors

E + S <-> ES -> E + P
E + I <-> EI
Ki = [E][I]/[EI]
• Competitive
• Uncompetitive
• Non-competitive
Types of Reversible Enzyme
Inhibitors
 Competitive Inhibitor (CI)

•CI binds free enzyme

• Competes with substrate for
enzyme binding.
•Raises Km without affecting Vmax
•Can relieve inhibition with more S
Competitive Inhibitors look like
substrate
O
O

HO C NH2 H2N S NH2

PABA Sulfanilamide
PABA precursor to folic acid in bacteria

O2C-CH2-CH2-CO2 -------> O2C-CH=CH-

CO2
succinate fumarate

Succinate dehydrogenase

O2C-CH2-CO2
 Uncompetitive Inhibitor (UI)

•UI binds ES complex

• Prevents ES from proceeding to E + P or back
to E + S.
• Lowers Km & Vmax, but ratio of Km/Vmax
remains the same
 Non-competitive Inhibitor (NI)

•NI can bind free E or ES complex

•Lowers Vmax, but Km remains the same
•NI’s don’t bind to S binding site therefore don’t effect
Km
• Alters conformation of enzyme to effect catalysis
but not substrate binding
 Regulation of Enzyme
Activity
1) Feedback inhibition: some enzymes are
inhibited by their own final product

E1 E2 E3
A↔B↔C↔D

Note: Here, the final product D inhibits the first

enzyme E1 and hence the whole reaction is
slowed down.
Eg: Conversion of L-threonine to L-Isoleucine
where the 1st enzyme threonine
dehydrogenase is inhibited by the final
 Regulation of
Enzyme….
2) Allosteric regulation: activity of some
enzymes is regulated by molecules other
than their substrates called allosteric
modifiers. Such molecules alter the
conformation of active sites of allosteric
enzymes.
– Such enzymes have another site different
from the active site in their folded structure
called the allosteric site.
Eg. Phosphofructokinase is allosterically
regulated by AMP, ADP, ATP and Citrate.
The first two activate it but the later two
 Regulation of
Enzyme…
3) Covalent modification: some
enzymes change their activities
through a reversible covalent
modification of their structure.
Eg: Phosphorylation:
dephosphorylation of fatty acid
synthase and glycogen
phosphorylase.
NB: Phosphorylated form of glycogen
phosphorylase is active but
phosphorylated form of glycogen
synthase is inactive.
 Regulation of
4) ZymogenEnzyme…
activation: Proteolytic
enzymes are produced in their inactive
precursor form called zymogens but
after losing part of their peptide they
become active enzymes.

Eg: Pepsinogen, Chymotrypsinogen and

Trypsinogen are inactive zymogen forms of
the active enzymes Pepsin, Chymotrypsin
and Trypsin.

Some proteins are also produced in their

inactive preprotein form like proinsulin.
 Regulation of
enzyme…
5) Induction or Repression of Enzyme
synthesis: Cells can regulate the amount of
enzyme present by altering the rate of
enzyme synthesis

The increased (induction) or decreased

(repression) of enzyme synthesis leads to
alteration in the total amount of active sites.

Induction and repression are slow processes

that may be elicited by hormone release. Eg.
Insulin increases the synthesis of key
 Enzymes in Clinical
Diagnosis
• Non functional plasma enzymes are
used extensively in clinical diagnosis.
• The concentrations of tissue and
plasma enzymes is maintained at
some levels under normal conditions.
• During tissue damage the
enzymes that were functional in
the tissue leak out increasing the
plasma enzyme concentration.
• This is used for diagnosis of organ
and tissue damages and
 Liver enzymes

• For the normal function test of the liver the typical

enzymes expresses in that organ are used and
these include:
 Serum Glutamate Pyruvate Transaminase (SGPT)
 Serum Glutamate Oxaloacetate Transaminase
(SGOT)
• Excess level of these enzymes in serum indicates
damage of liver cell (hepatitis, viral infections,
cirhosis etc)
77
 Heart diseases

• The heart isoenzymes of creatine kinase

(CK) and Lactate dehydrogenase (LDH) are
useful for diagnosis in addition to
Troponin measurement.
• Normally, the concentration of these
enzymes in serum is very low but when
heart cells get damages they leak into
the blood stream increasing the
concentration to a maximum.
 Muscle cell diseases

• The muscle isoenzymes of CK (MM) and lactate

dehydrogenase are used as marker enzymes
for the diagnosis of muscle cell associated
diseases like muscular dystrophy.
 Diseases related to bone

• Alkaline phosphatase is used as a key enzyme

for diagnosis of abnormalities in bone cells.
 Summar
• y factors that affect the rates of enzyme-
The study of enzyme kinetics—the
catalyzed reactions—reveals the individual steps by which enzymes transform
substrates into products.
• Keq, a ratio of reaction rate constants, may be calculated from the
concentrations of substrates and products at equilibrium or from the ratio
k1/k–1. Enzymes do not affect Keq.
• Measurement of the rate of an enzyme-catalyzed reaction generally employs
initial rate conditions
• Linear forms of the Michaelis-Menten equation simplify determination of Km
and Vmax
 Summar
• y be determined by using transformations of the
The values of Vmax and Km can
Michaelis-Menten equation
• The limiting rate of an enzyme-catalyzed reaction at saturation is described by
the constant kcat, the turnover number. The ratio kcat/Km provides a good
measure of catalytic efficiency.
• The effects of simple competitive inhibitors, which typically resemble
substrates, are overcome by raising the concentration of the substrate.
uncompetitive inhibitors lower Vmax and Km.
• Most metabolic control mechanisms target enzymes that catalyze an early,
committed, and rate-limiting reaction. Control can be exerted by varying the
concentration of the target protein, its functional efficiency, or some
combination of the two.
• Serum levels of enzymes are used for the diagnosis of organ and tissue
damage