PABZA

Assoc. Prof. Dr. Atif Amin Baig ,

Faculty of Medical Sciences,
University Sultan Zainal Abidin.
atifamin@unisza.edu.my
Recalls
To have understanding of:

1. What is a catalyst

2. What is the principle of catalyst action

3. Reaction kinetics

4. Activation energy

5. Reaction rate

6. Q10 Law

7. Reaction Order

8. What are enzymes?

9. Classification of enzymes?

10. Classes or enzymes and basis of nomenclature.

11. What are co-factors and coenzymes?

12. Basic characteristics of enzyme actions.

Learning Outcomes

 The relationship between reaction velocity and substrate

concentration of an enzyme catalyzed reaction.
 What is enzyme kinetics
 Substrate specificity and enzyme kinetics
 Michaelis-Menten equation
 Ping Pong Mechanism
 Factors effecting Enzyme Activity
 Enzyme Kinetics
Kinetos is a greek word meaning “moving”. Kinetics is
derived from kinetos.

 What it means? It means the Moving or Power or enzyme?

 Can we measure the “catalytic power”, “substrate specificity
and affinity” and its response towards “Enzyme inhibitors”?

 Using mathematical tool?

 Study of this analytical part of the enzyme is known as

enzyme kinetics.

 Study of the dependence of the reaction rate on the reaction conditions

Enzymatic Activity
 The catalytic action of an enzyme, its activity, is
measured by determining the increase in the
reaction rate under precisely defined conditions

 It is the difference between the turnover of the

catalyzed reaction and uncatalyzed reaction in a
specific time interval.
Enzymatic Activity
 Definition?

Reaction rates are expressed as the

change in concentration per unit of time
(mol/s–1)
Units of Enzymatic Activity

1. The catalytic activity of an enzyme is

independent of the volume, the unit used
for enzymes is usually turnover per unit
time, expressed in katal (kat, mol s–1)

2. international unit U is still more

commonly used (μmol turnover min–1; 1 U
= 16.7 nkat).
EA Vs Reaction & Substrate Specificity
 The action of enzymes is usually
very specific

 This applies not only to the type

of reaction being catalyzed, i.e.,
Reaction Specific OR Limited
Reaction.

 Also applies to the nature of the

reactants (“substrates”) that are
involved, i.e., Substrate Specific.

 Basis of classification of
enzymes?
Enzymatic Catalysis
 Enzymes are extremely effective catalysts.

 They can increase the rate of a catalyzed reaction by

a factor of 1012 or more

 Enzyme 1 (lecture)
 Un-Catalyzed Reaction

As a result of these limitations, conversion only happens

occasionally in the absence of a catalyst, and the reaction rate v
is low, even when the reaction is thermodynamically possible,
i.e., when ∆G < 0
Catalyzed Reaction
Catalyzed Reaction

 Exclusion of water
Catalyzed Reaction

 Active Site
Catalyzed Reaction

 Proximity & Orientation of Substrate

Catalyzed Reaction

 stabilization of the transition state

as a result of interactions between

the amino acid residues of the
protein and the substrate

reduces the activation energy

needed to create the transition
state

Catalysis
Catalyzed Reaction

 Formation of Product

as a result of interactions between

the amino acid residues of the
protein and the substrate

reduces the activation energy

needed to create the transition
state

Catalysis
Principle of Enzyme Catalysis
Principle of Enzyme Catalysis
 It is difficult to provide quantitative estimates of the
contributions made by individual catalytic effects

 Enzyme’s stabilization of the transition state is

the most important factor

 It is not tight binding of the substrate that is

important

 Enzyme would increase the activation energy

required by the reaction, rather than reducing it but
rather through the binding of the transition state.
 Reaction velocity and substrate concentration

Triose phosphate isomerase

Glyceraldehyde - 3 phosohate Dihydroxyacetone phosphate

Over the course of reaction, the concentration of the substrate falls as the
concentration of the product rises. The progress of this reaction or any other reaction
can be expressed as velocity (v); i.e, either the rate of disappearance of the substrate
(S) or the rate of appearance of the product (P)

d [ S ] d [ P]
v 
dt dt
Progress of the triose phosphate
isomerase reaction

Over time, the

concentration of the
substrate decreases
and the concentration
of the product
increases.
 Relationship between enzyme
concentration and reaction velocity

The more enzymes

present, the faster the
reaction.
[Enzyme] Vs [Substrate] Vs Reaction Rate

 When the enzyme concentration is held constant, the reaction

velocity varies with the substrate concentration, but in a nonlinear
fashion

 As small amounts of substrate are added to the enzyme

preparation, enzyme activity (measured as the reaction velocity)
increases almost linearly

 However, enzyme activity increases less dramatically as more

substrate is added

 Finally a point is reached beyond which enzyme activity appears to

level off as it approaches a maximum value with increase in
substrate
 [Enzyme] Vs [Substrate] Vs
Reaction Rate
 This plateau is known as maximum velocity (V max).

 This behavior shows that at low substrate

concentrations, the enzyme quickly converts all the
substrate to product

 But as more substrate is added, the enzyme becomes

saturated with substrate resulting in formation of a
hyperbola in the graph of a plot of reaction velocity
verses substrate concentration.

 The curve expressing this relationship has the same

general shape for most enzymes (it approaches a
rectangular hyperbola).
A plot of reaction velocity versus
substrate concentration

Here varying amounts of

substrate are added to a
fixed amount of enzyme.
The reaction velocity is
measured for each
substrate concentration
and plotted. The resulting
curve takes the form of a
hyperbola (a
mathematical function in
which the values initially
increase steeply but
eventually approach a
maximum level).
Michaelis-Menten equation
 The curve described in the previous
slide shows that:

ES complex is the key to understanding

this kinetic behavior.

In 1903, this kinetic pattern led Victor Henry to propose that an enzyme
combines with its substrate molecule to form ES complex as a necessary
step in enzyme catalysis.
Michaelis-Menten equation
In 1913, Leonor Michaelis and Maud Menten expanded
the idea of Victor Henry into a general theory of
enzyme actions. Michaelis and Menten postulated that:

- the enzyme first combines reversibly with its substrate

to form a ES complex in a relatively fast reversible step

- the ES complex then breaks down in a slower second

step(rate-limiting step) to yield the free enzyme and the
product
Michaelis-Menten equation
 Michaelis-Menten equation is the rate equation for an enzyme
catalyzed reaction and is the mathematical description of the
hyperbolic curve we have discussed earlier. The formula is

V max [ S ]
V0 
Km  [ S ]
Where,
V0 is the initial velocity
Vmax is the maximum velocity
[S] is the substrate concentration
Km (Michaelis-Menten constant) is the substrate concentration
at which the reaction velocity is the half of the maximum velocity.
Graphical determination of KM
Michaelis–Menten kinetics
Changes in concentration for a simple
enzyme-catalyzed reaction

ES remains constant
while S is converted to
P.Here all the
substrate is converted
to P
Lineweaver–Burk plot
Allosteric Vs Isosteric Enzymes?
efficiency of substrate binding (dashed curve)
declines constantly with increasing [A],
because the number of free binding sites is
constantly decreasing. In most allosteric
enzymes
binding efficiency initially rises with increasing [A], because
the free enzyme is present in a low-affinity conformation
(square symbols), which is gradually converted into a
higher-affinity form (round symbols) as a result of binding
with A.
 Factors Effecting Enzyme Kinetics
Energy levels of molecules

Activation energy
of uncatalysed
Initial energy state Activation energy reactions
of substrates of enzyme catalysed
reaction

Final energy state of

products

Progress of reaction (time)

1. Temperature
2. Hydrogen ion concentration(pH)
3. Substrate concentration
 Effect of Temperature
 Raising the temperature increases the rate of enzyme catalyzed
reaction by increasing kinetic energy of reacting molecules.

 Enzymes work maximum over a particular temperature known as

optimum temperature. Enzymes for humans generally exhibit
stability temperature up to 35-45 ᵒC.

 The temperature coefficient is a factor Q₁₀ by which the rate of

biological processes increases for a 10 ᵒC increase in temperature.
 For most biological processes Q₁₀ = 2.

 However some times heat energy can also increase kinetic energy
to a point that exceed the energy barrier which results in denaturing
of enzymes.
 5- 40oC Temperature
Increase in Activity

40oC - denatures

Rate of Reaction

0 10 20 30 40 50 60

<5oC - inactive
 Effect of pH
 Rate of almost all enzymes catalyzed reactions depends on
pH
 Most enzymes exhibit optimal activity at pH value between 5
and 9
 High or low pH value than optimum value will cause
ionization of enzyme which result in denaturation of enzyme
 pH affects the formation of hydrogen bonds and sulphur bridges
in proteins and so affects shape.

trypsin arginase
pepsin
Rate of Reaction (M)

Acidic 2 4 6 8 10
pH Basic
Substrate Specificity Pockets
Ping-Pong Mechanism
(Bi-substrate Kinetics)
Acknowledgments
 Dr. Meera Kaur
University of Winnipeg

Thank you for listening