Enzyme Kinetics

Using mathematical tool, we can describe an enzyme’s catalytic power, its substrate affinity, and its
response to inhibitors, etc.

– Study of this analytical part of the enzyme is known as enzyme kinetics.

WHY?
1. How fast an enzyme catalyzed reaction goes?
2. Why study enzyme kinetics?
– Helps us understand mechanism of enzyme (how it works)

– Investigation of mutations in metabolic pathways

– Understanding of different environmental factors affecting enzyme activity and regulation of biochemical
reactions (up or down regulation of catalyst).
2. Application in Biochemical Engineering?
– Helps us in calculating reaction time, yield and optimum economic condition required for the design of a
bioreactor
 k1
E  S  ES 
k2
E  P
k -1

• Total Enzyme concentration remains constant during a reaction.

• When amount of enzyme is very small as compared to amount of
substrate, the Enzyme-Substrate complex does not depletes the
substrate significantly.
• The product concentration is very low therefore, product inhibition
may be considered negligible.
 Triose Phosphate Isomerase

Glyceraldehyde - 3 phosphate 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) or the
rate of reaction (r); i.e. either the rate of disappearance of the substrate (S) or the rate of
appearance of the product (P).
Enzyme
Catalysed
reaction
Progress
• Over time, the
concentration of the
substrate decreases
and the
concentration of the
product increases.
 Enzyme
Concentration and
Reaction Velocity

• To understand the effectiveness and characteristics of enzyme it is

important to study the influence of substrate, product and enzyme
concentration on the reaction rate.
• Increase in enzyme concentration increases the velocity/speed of the
biochemical reaction. i.e. More Enzymes, more faster Reaction
 Substrate 1. At low substrate concentration, the reaction rate
Concentration and is proportional to the substrate concentration
Reaction Velocity or (first order reaction).
rate of reaction
2. At higher substrate concentration, reaction rate is
independent of substrate and the reaction rate
follows the first order to zero order reaction.

3. Maximum reaction rate rmax is proportional to the

enzyme concentration within the range of
enzyme tested.
Conditions for Michaelis -Menten
Two assumptions must be met for the Michaelis-Menten equation
·Equilibrium -the association and dissociation of the substrate and
enzyme is assumed to be a rapid equilibrium and Ks is the
enzyme:substrate dissociation constant.
• Steady state - the enzyme substrate complex ES is at a constant value.
That is the ES is formed as fast as the enzyme releases the product.
For this to happen the concentration of substrate has to be much higher
than the enzyme concentration. That is why we only study the initial
velocity. Later in the reaction the substrate concentration is relatively
lower and the rate of product starts to be limited by diffusion and not
the mechanism of the enzyme.
 Michaelis-Menten equation
•  The total molar concentration (kmol/m-3) of free and combined
enzyme-substrate complex should be constant
k
S+E 1
k
ES…..…eq. 1
2

ES k 3
E + P……..eq. 2
CE0 = CES + CE …………eq. 3
• If reaction rate is slower, the rate of substrate consumption and rate
of product formation is equal to ES complex.
rate or = k3 [ES]
According to eq.1 rate of forward reaction is equal to rate of reverse
reaction.
k1[S][E] = k2 [ES]
• 
• The rate equation is called the Michaelis-Menten equation

• This equation can also be written as:

Vmax is the initial reaction velocity when [S] >> [Etotal].

Km is the Michaelis constant and is the concentration of substrate

resulting in half maximal velocity i.e. when [S] >> [Etotal. Unit of Km is
same as that of the substrate.
• 
PHYSICAL MEANING OF KM
• From the plot, Velocity is half maximum
when [S] = KM

• Substituting KM for [S] we get,

Km Tells us:
1. Max Amount of substrate
molecules per active site per
Km is a property associated with binding of S to E, second
2. Turnover Number
not a property of turnover.
PHYSICAL MEANING OF Vmax
• Vmax is the reaction velocity when the substrate concentration is high
compared to Km:
If [S] >> Km then V0 = Vmax

• This result follows directly from Michaelis-Menten equation:

if [S] >> Km, then [S] + Km ~ [S],

so [S]/(Km+[S]) = 1,

and V0 = Vmax.
 Kcat or K2 and Physical meaning of kcat
• Rate of catalysis is the maximum velocity independent of how much enzyme we
originally add in the reaction
• That would be kcat = Vmax / [E]tot
k1
E  S  ES 
k2
E  P
k -1

Physical Meaning of kcat?

• kcat is the rate of rate-limiting step in the equation i.e. k2 or kcat= k2
• Describes turnover of substrate to product:
Number of product molecules produced per sec per molecule of enzyme
• More complex reactions may not have kcat = k2, but we can often approximate them
that way anyway
• Some enzymes are very efficient: kcat > 106 s-1
What do Vmax and kM tell us?
• Vmax and kM are properties associated with the enzyme’s ability to turn over substrate
and its ability to bind substrate respectively.

• kM tells us the affinity of the substrate and Vmax relates to efficiency

• KM tell how much substrate to use in an assay

• If more than one enzyme share the same substrate, KM will also determine how to
decide which pathway the substrate will take

• Vmax or kcat tells about rate limiting enzyme in pathway, and used to determine the
effectiveness of inhibitors and activators.