• Enzymes are most remarkable and highly specialized proteins (with the exception of few

RNAs) that act as reaction catalysts of biological systems.

• These are central to every biochemical process.

• Acting in organized sequences, they catalyze the hundreds of stepwise reactions that-

 degrade nutrient molecules,

 conserve and transform chemical energy, and

 make biological macromolecules from simple precursors

• Enzymes are highly effective catalysts, commonly enhancing reaction rates by a factor of 105 to

1017.

• Many enzymes require non-protein coenzymes or cofactors for their catalytic function.
• The function of enzymes and other catalysts is to lower the activation energy, G ‡, for a reaction
and thereby enhance the reaction rate.
• The equilibrium of a reaction is unaffected by the enzyme.
 EnzymE KinEtics
• The study of the rates of chemical reaction is called Kinetics and the study of the rates of enzyme-
catalyzed reactions is called Enzyme kinetics.
• It refers to determination of rate of a reaction and how it changes in response to changes in
experimental parameters.
• Kinetic measurements of enzymatically catalyzed reactions are among the most powerful techniques
for elucidating the catalytic mechanisms of enzymes.
• One simplifying approach in kinetics experiments is to measure the initial rate or initial velocity V0
which is measured by studying substrate saturation curve.
• The graph is plotted between substrate concentration and velocity (V0).
• The amount of enzyme is constant, and the velocity of the reaction is determined at various substrate
concentrations. The reaction rate, v, as a function of [S] is described mathematically by a rectangular
hyperbola.
• This content should be on blank page not on ruled page.
• The Graph shows that-
 At low concentrations of the substrate S, v is proportional to [S] (First order kinetics)
 At high [S], v becomes virtually independent of [S] and approaches a maximal limit- Vmax (zero-order
kinetics)

Substrate saturation curve for an

enzyme-catalyzed reaction
 MICHAELIS-MENTEN KINETICS
• Leonor Michaelis and Maud L. Menten proposed a general theory of enzyme action in 1913 consistent with
observed enzyme kinetics.
• They proposed a simple model that accounts for most of the features of enzyme-catalyzed reactions.
• In this model, the enzyme reversibly combines with its substrate to form an ES complex that subsequently yields
product, regenerating the free enzyme.

S -is the substrate

E- is the enzyme
ES- is the enzyme–substrate complex
P- is the product
k1, k-1, and k2 are rate constants
• The Michaelis-Menten equation describes how reaction velocity varies with substrate
concentration

Km = Michaelis constant = (k-1 + k2)/k1

Characteristics of Km: Km, the Michaelis constant, is characteristic of an enzyme and its particular

substrate and reflects the affinity of the enzyme for that substrate.

• Km is numerically equal to the substrate concentration at which the reaction velocity is equal to

1⁄2Vmax.

• Km does not vary with enzyme concentration.

• if 1/v o is plotted versus 1/[S], a straight line is obtained.

• This plot, the Lineweaver-Burk plot (also called a double reciprocal plot) can be used to calculate

more accurate values of Km and Vmax

y= 1/V0
m= km/Vmax
b=1/Vmax
 A double-reciprocal or Lineweaver-Burk plot.

This line has a slope of Km/Vmax, an intercept of 1/Vmax on the 1/V0 axis, and an intercept of
1/Km on the 1/[S] axis.
• It is equivalent to the number of substrate molecules converted to product in a given unit of time on a
single enzyme molecule when the enzyme is saturated with substrate.
• Kcat is equivalent to the rate constant of the rate limiting step of the reaction.

 Enzyme first combines reversibly with its substrate to form an enzyme-substrate 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 reaction product P Hence Kcat= K2.
• When several steps are partially rate-limiting, kcat can become a complex function of several of the
rate constants that define each individual reaction step.
 • Putting the valve of Vmax the MM equation now becomes

• The Ratio, kcat /Km, Defines the Catalytic Efficiency of an Enzyme

As Km is inversely proportional to the affinity of the enzyme for its substrate and kcat is directly

proportional to the kinetic efficiency of the enzyme, kcat/Km provides an index of the catalytic efficiency

of an enzyme operating at substrate concentrations substantially below saturation amounts.

numEricals
1. Estimation of Vmax and Km by Inspection-
Estimate the Vmax and Km of the enzyme-catalyzed reaction for which the following
data were obtained.
2.
3.
4.
5. The following experimental data were collected during a study of the catalytic activity of an intestinal
peptidase with the substrate glycylglycine:
Glycylglycine +H2O 2glycine

Determine the Km and Vmax for this enzyme preparation and substrate using double reciprocal plot.
6.
7.

Hint: See theory how Kcat , Vmax and Et (total enzyme) are related.
8. Applying the Michaelis-Menten Equation a research group discovers a new version of hexokinase,
which they call hexokinase*, that catalyzes the chemical reaction
Glucose to Glucose 6- phosphate
The researchers begin to characterize the enzyme.
(a) In the first experiment, with [Et] at 4 nM, they find that the Vmax is 1.6 micro molar per sec. Based on this
experiment, what is the kcat for hexokinase*?
(b) In another experiment , with [Et] at 1nm and [glucose] at 30 micro molar V0 is 300 nm per sec. Calculate
Km?
Note (please answer in appropriate units)
9. α- chymotrypsin from bovine pancrease catalyzes the hydrolysis of N-acetyl-L- phenylalanine p-
nitrophenyl ester. The experimental data for the initial rate as a function of substrate concentration are
given. Determine the Km and V max with the help of Michaelis -Menten graph .