ENZYME KINETICS

• The study of the rates of chemical reactions is called kinetics

• The study of the rates of enzyme-catalyzed reactions is called enzyme kinetics

• Kinetic description of enzyme activity will help to understand how enzymes function.

• At what rate do enzymes react? How does rate change with varied conditions?

• To study the mechanism of an enzyme-catalyzed reaction by determining the rate of enzyme

reaction and how it changes in response to changes in experimental parameters

• Enzyme kinetics addresses the biological roles of enzymatic catalysts and quantify the remarkable

function of biological enzymes;

• Enzyme kinetics information can be exploited to control and manipulate the course of metabolic

events.
• Expression for enzyme catalyzed reaction:

k1 k2
E+S ES E+P
k-1
 Michaelis-Menten Equation
• At very low substrate concentration, when [S] is much less than KM,
V0 = Vmax[S] / KM + [S]
• Rate increase with [S]
• Rate levels off as approaches Vmax
• More S than active sites in E, Adding S has no effect
• At V0 = ½ Vmax
• [S] = KM
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 Significance of Km
• The substrate concentration at which enzyme-catalyzed reaction proceeds at one-half
of its maximum velocity

• Km is independent of [E]. It is determined by the structure of E, the substrate and

environmental conditions (pH, T, ionic strength, …)

• Km is the [S] at 1/2 Vmax

• Km is a constant for a given enzyme

• Small Km means tight binding; high Km means weak binding

• Km is equal to the concentration of substrate at which half the active sites are filled.

Thus, Km provides a measure of the substrate concentration required for significant

Analysis of Kinetics

 When [S] << KM, the reaction increases

linearly with [S]; I.e. vo = (Vmax / KM ) [S]
 When [S] = KM, vo = Vmax /2
 When [S] >> Km, vo = Vmax
V0

Vmax

Vmax/2

0 [S]
Km
• The value of Km quantifies the affinity of the enzyme and the substrate.

• The larger the Km the smaller the affinity.

• The magnitude of Km varies widely with the identity of the enzyme and the nature
of the substrate. It is also a function of temperature and pH.
A small Km indicates that the enzyme requires only a small amount of
substrate to become saturated. Hence, the maximum velocity is reached at
relatively low substrate concentrations. A large Km indicates the need for high
substrate concentrations to achieve maximum reaction velocity.

• A measure of the substrate concentration required for effective catalysis to occur

 Km for selected enzymes
Enzyme Substrate km

Catalase H2O2 25

Hexokinase ATP 0.4

D-Glucose 0.05

D-Fructose 1.5

Carbonic anhydrase HCO3- 9

Chymotrypsin Glycyltyrosinylglycine 108

N-Benzoyltyrosinamide 2.5

Galactosidase D-Lactose 4

Threonine dehydratase L-Threonine 5

 that determines the amount of product and overall regulation of a given pathway. For
example :

• Clinically, Km comparisons are useful for evaluating the effects mutations have on protein

function for some inherited genetic diseases.

 Significance of Vmax

• The reaction velocity of an enzymatic reaction when the binding sites of E

are saturated with substrates.
• It is proportional to [E].
• The values of Vmax will vary widely for different enzymes and can be used as
an indicator of an enzymes catalytic efficiency. It does not find much clinical
use
• Vmax is a constant with units s-1
• • Vmax is the theoretical maximal rate of the reaction –
• • To reach Vmax would require that all enzyme molecules are tightly bound
with substrate
• • Vmax is asymptotically approached as [S] is increased
 Catalytic Constant kcat
kcat = Vmax / [E]T
• Turnover number
• Number of reaction processes each active site catalyzes per unit time
• Measure of how quickly an enzyme can catalyze a specific reaction
• A measure of catalytic activity
• The kcat is a direct measure of the catalytic production of product under
saturating substrate conditions.
• the maximum number of substrate molecules converted to product per enzyme
• molecule per unit of time.
 Catalyticefficiency
Kcat / KM
• Specificity constant
• by using Kcat/KM values, we can compare an enzyme’s preference for
different substrates..
• kcat and Km are useful for the study and comparison of different enzymes
• It is the Kcat/KM value that allows direct comparison of the effectiveness of
an enzyme toward different substrates.
• Different enzymes might have similar efficiencies but very different Km
and Kcat values
• it takes into account both the rate of catalysis with a particular substrate
(kcat) and the nature of the enzyme–substrate interaction (KM)
The most efficient enzymes have kcat/KM values with upper limit of 108 to 109 M-1.s-1
Lineweaver-Burke transformation of the
Michaelis-Menton equation.

• experimentally determine Km and Vmax

• Used for Interpreting Vmax and Km
• ,Helps to understand how Km and Vmax parameters enhance
understanding of enzyme-catalyzed reactions
• Velocity vs substrate plots are useful for visually estimating kinetic
parameters
• Hyperbolic curves cannot properly determine the upper limit of the curve
(Vmax)
• Transforming the data to a form that can be plotted as a line.
 Lineweaver-Burk plot

1 Km 1 1
= +
V Vmax [S] Vmax

• To determine Km and Vmax

Lineweaver-Burk Plot
• Method to determine Vmax and KM
• Reciprocal of M-M equation
• Linear equation

1 K M  [S] K M 1 1
  
V0 Vmax [S] Vmax [S] Vmax