Chapter 2

Catalytic Principles and Reaction

Mechanisms of Enzymes
 How Enzymes Work
• An enzyme provides a specific env`t within which a given
reaction can occur more rapidly.
• The distinguishing feature of an enzyme-catalyzed reaction
is that it takes place within the confines of a pocket on the
enzyme
• active site
• The molecule that is bound in the active site and acted upon
by the enzyme
• substrate
 ENZYME ACTION – can occur two ways:
• Lock and Key model – the substrate molecule
has a specific 3-dimensional shape that allows it
to fit into the specific 3-dimensional shape of an
enzyme’s active site.
• Both enzyme and substrate already exist in these
specific 3-dimensional shapes.
• Induced Fit model – An interaction b/n the
enzyme and substrate induces or changes the ENZYME
shape of the molecules to produce a suitable fit.
 How enzymes work

• Enzymes
• Catalyze a reaction until it reach equilibrium
• Does not change the equilibrium
• Its role is to increase the reaction rate

• All reactions in living organisms

• Catalyzed by enzymes
• Uncatalyzed reactions take time to reach equilibrium
 Chemical reactions
• The rate of a chemical reaction depends on the activation energy.

• Minimum quantity of energy which the reacting species must posses in order to
undergo a specified reaction.

• The potential energy of the system as a chemical change occurs can be plotted vs.
time:
• The starting point for either the forward or the reverse reaction is called the ground
state.
• Ground state - the lowest possible energy that a molecule has.
• The d/nce b/n the energy levels of the ground state and the transition state is the
activation energy, G‡.
• a higher activation energy corresponds to a slower reaction.
 Chemical reaction

• Reactants must collide

• Product formation
• Barriers
• Repulsion(steric repulsion)
• Sufficient energy
• Right orientation
• Solution
• Increase temp
• Increase concentration /
• Pressure for gasses
• Energy barrier
• Activation energy, Ea
 Chemical reactions …
• Source of energy
• Collision –successful collision
• The colliding molecules must have
• Sufficient energy
• Be in the right orientation
• Hit with sufficient force
• More molecules have large kinetic energies at high
temp than at low temp
 Mechanism of enzyme reaction
• Rate of a chemical reaction depends on the activation energy
• The activation energy can be calculated by taking the difference of the transition state enthalpy and
the reactant enthalpy (ΔH). 
• Higher Ea –  slower reaction rate
• ΔH (enthalpy of the rxn)
• potential energy of reactants and products
• products have less energy than reactants
• Example - energy of reactants = 180 kcal/mol
- energy of products = 80 kcal/mol
ΔH = 80- 180kcal/mol = 80kcal/mol

• For exothermic rxns- heat is released to the outside

and ΔH is negative

• Energy obtained to pass the Ea barrier is from collisions

 Mechanism of enzyme reaction ...
Maxwell-Boltzmann Distribution
• How much energy needed to put in system for a chemical reaction to occur
• At high temp
• molecules have large kinetic energy
• there will be more energy in the reactants – with more energy they move faster
• makes them more likely to collide causing chemical reaction
• rate increases with rising temp
• Activation energy will remain
the same
• curve shifted to the right
 The role of catalysts…
• For a reaction to occur reactant molecules must contain sufficient
energy to cross a potential energy barrier- the activation energy
• All catalysts lower activation energy
• Form a transition state with the reactants
E+S ES EP E +P
Rate limiting step

 Rate limiting step - affect metabolic pathways at the earliest step

- product formation step
ΔG = ΔH - TΔS

• Gibbs free energy, denoted

ΔG, combines enthalpy
and entropy into a single
value. The change in free
energy, ΔG, is equal to the (Ground state for reactants)
sum of the enthalpy, ΔH plus
the product of the temperature,
T and entropy, ΔS of the
system.
• Enthalpy is the measure of total heat
present in the thermodynamic (Ground state for products)
system.
• Entropy is the measure of disorder
in a thermodynamic system.
 How the activation energy decreases ?
• Enzyme bind the substrate(s)
• Correct orientation to react
• Close to the catalytic groups on the active enzyme complex and any other substrate
• Introduction of strain into the reactants
• Provision of an alternative reactive pathway
• Binding
• Structural complementarity
• Specificity
• Minimal steric repulsion
• Absence of unsolvated or unpaired charges – presence of unpaired and charged
donors/acceptor weakens binding
• The presence of sufficient hydrogen bonds - essential in determining the structure of
proteins and nucleic acids; it is a major determinant of specificity in enzyme catalysis
• Hydrophobic or hydrophilic nature of substrates
 Enzyme catalysis and active site
• All reactions in living cells
catalyzed by enzymes
• Catalytic machinery at the
active site
• Protein 3D structure
• Ribbon diagram of bovine chymotrypsin (EC 3.4.21.1)
in a complex with the transition-state analog
inhibitor N-acetyl-L-leucyl-L-phenylalanine
trifluoromethylketone (AcLF-TFK)
• The ball and stick model is the inhibitor
• The gray ball and sticks are amino acid chains of the
catalytic residues of Ser 192,His 57, Asp102
 Conformational mobility and catalysis

•  Hexokinase
it transfer the phosphate group
to H-OH

Glucose + MgATP G–6-P + MgADP

• Yeast hexokinase
• Substrate-induced conformational change
• Binding of glucose induce conformational
change
• Catalyze the P transfer from MgATP exclusively
to the C6(OH) of glucose
• Binding of glucose lead to the closure of a cleft

Saccharomyces cerevisiae hexokinase (EC 2.7.1.1)

with glucose (black ball-and-stick representation)
bound in the active site cleft. The N-terminal domains
of the Cα traces of the liganded- (gray, PDB 1HKG, PI
isozyme) and apo- (red, PDB 2YHX, PII isozyme)
hexokinase structures are aligned to show the apparent
motion of the C-terminal half of the enzyme as the
whole protein contracts around the substrate
 Structural mobility
• Enzymes
•  Structurally dynamic molecules
•  Can adapt to changing catalytic requirements
• Movement
•  Allow the transient opening of gaps and
•  Expose side chains in the α- helices and β-
sheets to external molecules
• No disruption of 20 structure
• Structural mobility
•  Important for enzyme specificity and
catalysis of multistep reactions Motion of helices and domains in proteins
 Reaction progress
• Progress curves vary depending on
• Medium pH
• Optimum temperature
• Ionic strength
• Polarity
• Substrate type, and
• Enzyme and coenzyme concentration
• Minimum substrate concentration
to have conversion ~20%
• Reaction velocity decreases in time
• As the enzyme molecules become
saturated with substrate, this increase
in reaction rate levels off. 
• Enzyme becomes unstable
• Degree of saturation of the enzyme
decreases as substrate is depleted
• Reverse reaction becomes more
predominant
• Inhibition by the products
• Any combination of the above
Reaction progress
 To determine reaction velocity, v

• A tangent to the progress curve is

drawn
• Slope of this tangent (i.e., using linear
regression) = V
 Enzyme kinetics and Michaelis-Menten constants

• Reaction rate in the presence and absence of enzymes.

• For enzyme catalyzed reaction, curve is hyperbolic.
 Enzyme kinetics

• Rate of reaction with increasing substrate concentration.

 Vo = d[P] = Vmax [S]
dt [S] + Km
The Michaelis-Menten equation arises from the general equation for an enzymatic reaction :
E +S K1
ES K2
E+P
k­‐1 k­‐2

• Where- E-enzyme, S- substrate, ES- enzyme- substrate complex and P- product

k1, k-1 and k2,k-2 - rate constants
• Association of enzyme and substrate (first step) is fast, while the catalytic (second step) is much
lower and is rate limiting, [ES] may dissolve back into the enzyme and substrate or move
forward to form product.
• At initial reaction time, when t = 0, little product formation occurs, therefore the backward
reaction, K-2 may be neglected.
• And the new reaction becomes
E+S K1
ES K2
E+P
K-1

• Assuming the steady state the following rate equation may be written as:
• Rate of formation of ES, ES = K1 [E] [S] The bracket represents concentration

• Rate of breakdown of ES, ES = (K-1+ K2) [ES]

• Rate of formation of ES = Rate of breakdown of ES
K1 [E] [S] = (K-1+ K2) [ES]

• Rearranging them [E] [S] = (K-1+ K2)

[ES] K1
• The fraction [E] [S] has been coined as Km Michaelis -Menten constant
[ES]
• According to Michaelis -Menten kinetics, at low concentration of substrate
[S], the concentration is almost negligible in the denominator as Km >>>
[S], [S] + Km ≈ Km, so the equation is essentially
Vo = Vmax [S]
Km
In this case the rate and the substrate concentration are directly proportional to
each other. The reaction is first-order kinetics
• At high substrate concentration [S], [S] >>> Km, Enzyme is saturated with
substrate, [S] + Km ≈ [S],the term
[S] becomes essentially one and the initial velocity (Vo) approaches to maximum
[S] + Km velocity (Vmax).

Vo = Vmax
• The velocity and is independent of the substrate concentration. The reaction is
zero-order kinetics.
• The rate of change [ES] equals
• the rate of its formation minus its breakdown (forwards to give
product or backwards to regenerate substrate)

• d[ES]/dt = K1[E][S] – (k-1 + k+2)[ES]

• E.g., At what substrate concentration does an enzyme reaches 80% of its Vmax?

V
o = 80% Vmax = 0.8 Vmax

= Vmax [S]
Vo
Km + [S]

0.8 Vmax = Vmax [S]

Km + [S]

0.8 Vmax = Vmax [S]X 1

( Vmax ) (Km + [S]) (Vmax)

0.8 Km +0.8 [S] = [S]

0.8 Km
= [S] - 0.8 [S]
Write the relationship that exists b/n Vmax, Km and Kcat (turnover
number) ?

• Total enzyme exist as

• free enzyme ([E]) or
• as ES complex ([ES])

• [ET] = [E] + [ES] where [ET] – total concentration of enzyme

 Michaelis constant, Km

• Michaelis Constant (Km): Enzymes have varying tendencies to bind their

substrates (affinities) - indicate binding strength (i.e. weak or strong binding.
• An enzyme's Km describes the substrate concentration at which half the
enzyme's active sites are occupied by substrate.
• [S]concentration, Km = ½ Vmax
• Km is a ratio of rate constants (K-1 +K2)
K1
• High Km indicates that the enzyme has low affinity for a substrate because the
enzyme is saturated with the high substrate concentration.
• On the other hand, a low Km means a high amount of substrate is needed to
saturate the enzyme, indicating a high affinity for substrate.
Michaelis—Menten plot
Initial velocity for a model enzyme
 Vmax

• Vmax
• The number of substrate molecules converted into product per unit time
• The rate of reaction when the enzyme is saturated with substrate
• Maximum rate of reaction
• At very high substrate concentration ([S] >> [ET]) for which all the enzyme
would be bound up with substrate (i.e., the enzyme is completely saturated with
substrate)
• [S] >> Km
• Vmax = Vo = k2[ES]
• Also at Vmax, [ES] = [ET]
• Vmax = Vo = k2[ES] = k2[ET]
 Kcat - turnover number

Kcat - turnover number

• The maximum number of substrate molecules that the enzyme can
'turnover' to product per unit time – turnover of substrate into product
• These are properties associated with the enzyme’s ability to turnover
substrate, not its ability to bind substrate.
• Kcat substituted for k2
Vmax = k2[ET]
Vmax = kcat[ET]
Example: Determine the initial rate of an enzyme, ET when conditions are such that the substrate concentration used is enough to produce half of E T`s

maximum rate, 40 μmol/sec. If the enzyme concentration is 10 mmol, determine the Kcat.

Solution: [S] at ½ Vmax is Km= [S]

Vo = ½ Vmax

Vo = Vmax [S] = Vmax [S] = Vmax [S] = 1/2 Vmax

Km +[S] [S] +[S] 2[S]

Vo = 1/2 Vmax = ½ ( 40 μmol/sec) = 20 μmol/sec

Vmax = Kcat[ET]

Vmax = Kcat[ET]

[ E T] [ET]

Kcat = Vmax = (40 μmol/sec) = (40 μmol/sec) = 4x 10-3/ Sec

[ E T] (10 m mol ) (10 x 103μmol )

 Specificity constant - Kcat/Km
• Determines enzyme`s catalytic efficiency.
• Determines the relative rate of reaction at low substrate concentration.
• Measures affinity of enzyme for a specific substrate.
• We call it the specificity constant for the enzyme for that particular substrate

• Useful in comparing primary substrate to other substrates (e.g., ethanol vs. propanol in
alcohol dehydrogenase)
• At low substrate conc ([S] << KM) the enzymatic rate is much less than kcat because
most of the active sites are unoccupied
vo = Kcat x [ET][S] - how it is derived ?
Km
• By combining the following equations

Vo = K2 [ES] = V = Kcat [ES] and [ES] = [ET] [S]

Km
• we get

Vo = Kcat x [ET][S]
Km
 Physical factors affecting enzyme activity
• Effect of pH
• Activity
• Stability
• Each enzyme has:
• Characteristic pH
• Narrow range (5-9) mostly
A variety of amino acid residues as well as the carboxyl and amide termini of proteins
have a pKa range in the range of intracellular pH.
As a result, a change in pH can protonate or deprotonate a side group, thereby changing
its chemical features.
• pH optimum
 Extremely high or low pH values generally result in complete loss of activity for most enzymes
• pH effect
• change charge distribution on the surface
• structural stability and solubility
• reactivity of the catalytically active groups around the active site
• pKa (acid dissociation constant)
• defined as -Log10(Ka) Ka - an acid dissociation constant
the lower the pH, the higher the concentration of hydrogen ions, [H+]
the lower the pKa, the stronger the acid and the greater its ability to
donate protons
• the pH at which half the groups are ionized, pH= pKa,
e.g., half of the amine groups are ionized at pH = pKa of amine. pKa of
amine = 9.69 so half of the amino group is ionized at pH = 9.69.
• pH change affect
• charge distribution on the substrate(s)
• product(s), and
• coenzymes
• Increasing [H+]
• increase competition of hydrogen ions for any metal cationic binding sites on the enzyme,
• reduce the bound metal cation concentration
• Decreasing [H+]
• increase in hydroxyl ion concentration
• Compete against the enzymes' ligands for divalent and trivalent cations
• cause their conversion to hydroxides
• at high hydroxyl concentrations, lead to complete removal from the enzyme.
 Charge variations

•Charge variations
Enzymes are amphoteric molecules containing a large number of acid and basic
groups, mainly situated on their surface.
The charges on these groups will vary, according to their acid dissociation
constants, with the pH of their environment.
This will affect the total net charge of the enzymes and the distribution of charge
on their exterior surfaces, in addition to the reactivity of the catalytically active
groups.
These effects are especially important in the neighbourhood of the active sites.
Taken together, the changes in charges with pH affect the activity, structural
stability and solubility of the enzyme.
Affect enzyme substrate binding and catalytic efficiency
 Ionic strength

• Ionic strength is a measure of the concentration of charges in a solution.

• The ionic strength may also affect the activity of an enzyme by changing the
stability and solubilities of the enzyme (charge on the active site of enzyme) as
well as those of the substrates (charge of substrate).
• The effects of salts on stability becomes more important with more hydrophilic
enzymes.
• If the charges are opposite then there is a decrease in the reaction rate with
increasing ionic strength whereas if the charges are identical, an increase in the
reaction rate will occur (e.g., the rate controlling step in the catalytic
mechanism of chymotrypsin involves the approach of two positively charged
groups, 57HIS+ and 145ARG+ causing a significant increase in kcat on
increasing the ionic strength of the solution.
 Temperature

• Temperature profile
• Increase in rate
upon increasing the temp from 20 to 40°C, an almost
threefold increase of Vo is observed.
•Affect weak bonds
•Denaturation
•Stability!!!
• Rates of all reactions, including those catalyzed by enzymes, rise with increase in
temperature in accordance with the Arrhenius equation.
K=Ae-Ea/RT
Where
• K - kinetic rate constant for the reaction,
• A - Arrhenius constant (the frequency factor) - the pre-exponential factor, a
constant for each chemical reaction. According to collision theory, A is the
frequency of collisions in the correct orientation
• Ea - activation energy  (kJ M-1)
• R – universal gas law constant
• T - absolute temperature (in Kelvin)
ln K = ln A -Ea/RT
 Two-Point Form of the Arrhenius Equation
• The activation energy can also be found algebraically by substituting two rate
constants (K1, K2) and the two corresponding reaction temperatures (T1, T2) into
the Arrhenius Equation
• lnK1 = lnA -Ea/RT1………………………….. *

• ln K2 = ln A -Ea/RT2……………………………**
• Subtracting equation (**) from equation (*) results in
ln K1 = Ea (1 - 1)………………………...............***
K2 R (T2 T1)
• Rearranging equation (***) Ea = RT1T2 ln k 1
(T2- T1) K2
• The rate constant for the reaction H2(g)  + I2(g) ---> 2HI(g) is 5.4 x 10-4 M-1s-1 at
326oC. At 410oC the rate constant was found to be 2.8 x 10-2 M-1s-1.

Calculate the:
a) activation energy and
b) Arrhenius constant for this reaction
T1= 326oC = 599K
T2= 410 oC = 683
R= 8.314 J/K mol
K1= 5.4 x 10-4 M-1s -1
K2 = 2.8 x 10-2 M-1s -1
ln k1 = Ea (1 - 1)
K2 R (T2 T1)

ln(5.4 x 10-4 M-1s -1/ 2.8 x 10-2 M-1s-1) = (-Ea /R ){1/599 K - 1/683 K}

-3.9484 = - Ea/R {2.053 x 10-4 K-1}

Ea= (1.923 x 104 K) (8.314 J/K mol)

Ea= 1.60 x 105 J/mol

 k=Ae-Ea/RT
K= 5.4 x 10-4 M -1s-1
Ea = 1.60 x 105 J/mol
T= 326oC = 599K
R= 8.314 J/Kmol
A= ???

{-(1.60 x 105 J/mol)/((8.314 J/Kmol)(599K))}

5.4 x 10 M s =A e
-4  -1 -1

A= (5.4 x 10-4 M-1s-1) / (1.141x10-14) = 4.73 x 1010 M-1s-1

 Storage
• In order to minimize loss of activity on storage, even moderate
temperatures should be avoided.
• Most enzymes are stable for months if refrigerated (0 – 4oC).
• Cooling below 0oC, in the presence of additives (e.g., glycerol) which
prevent freezing, can generally increase this storage stability even further.
• Freezing enzyme solutions is best avoided as it often causes denaturation
due to the stress and pH variation caused by ice-crystal formation.
• Other factors, such as the presence of thiol anti-oxidants, may improve
the thermal stability in particular cases. 
• It has been found that the heat denaturation of enzymes is primarily due to
the proteins' interactions with the aqueous environment.

• They are generally more stable in concentrated, rather than dilute, solutions.

• In a dry or predominantly dehydrated state, they remain active for long

period even at temperatures above 100oC.

• This property has great technological significance and is currently being

exploited by the use of organic solvents.
 Effect of pressure

• Affect enzyme reaction

• Upon increasing the pressure from 1 to 2000 bar,
Vo increases by 40%
• Mainly due to increase in collision
• Increase Kcat, how ?
• Decrease Km, how ?