KINETICS OF ENZYMATIC REACTIONS

Enzyme kinetics studies the influence of the chemical nature of the reactants (enzymes, substrates) and the conditions of their interaction (concentration, pH, temperature, presence of activators or inhibitors) on the enzymatic reaction. Enzymatic reaction rate (V) is measured by the loss of substrate or product gains per unit of time.

Enzyme (E) is reversibly connected to the substrate (S), forming an unstable enzyme-substrate complex (ES), which falls at the end of the reaction with the release of the enzyme (E) and the reaction products (P):

Saturation of the enzyme with the substrate is an important feature of enzyme reactions. At low substrate concentration the reaction rate is directly proportional to its concentration. At high substrate concentration the reaction velocity reaches a maximum (V_max). In this phase the velocity is constant and independent of substrate concentration and completely determined by the concentration of the enzyme (fig. 11).

Fig. 11. Dependence of the enzymatic reaction velocity on the substrate concentration at constant enzyme concentration.

The dissociation constant K_S of enzyme-substrate complex ES is the inverse of the equilibrium constant:

Lower value of K_s corresponds to the higher affinity of the enzyme to the substrate.

L. Michaelis and M. Menten proposed an equation, which is called the equation of Michaelis -Menten. It expresses the quantitative relationship between substrate concentration and the velocity of enzymatic reactions:

u is a velocity, V_max - a maximum velocity of enzymatic reactions.

Briggs and Haldane improved the equation by introducing the Michaelis constant K_m which is determined experimentally.

Briggs - Haldane equation:

Michaelis constant is defined as the substrate concentration to produce half-maximum velocity (fig. 12). K_m indicates the affinity of the enzyme to the substrate. The lower value corresponds to the greater affinity. The experimental values of K_m for the majority of enzymatic reactions usually are 10^-2-10^-5 M. If the reaction is reversible, the interaction of the enzyme and substrate in forward reaction is characterized by direct K_m, which differs from that of the reverse reaction.

Fig. 12. Graphical determination of the Michaelis constant.

 

ENZYME PROPERTIES

Enzymes differ from the usual catalysts with a number of properties.

Heat-labile or heat-sensitive (fig. 13).

Fig. 13. Dependence of the enzymatic reaction velocity on temperature.

 

At temperatures below 45-50°C, the velocity of most biochemical reactions is increased twice when the temperature increase of 10°C (Vant Hoff's rule). At temperatures above 50°C a heat denaturation of the enzyme begins. It is greatly influenced the reaction rate and can lead to a complete cessation of the enzymatic process.

The temperature, at which the catalytic activity of the enzyme is maximal, is called its temperature optimum. The temperature optimum for most enzymes of mammals is in the range 37-40°C. At low temperatures (0°C and below) enzymes are usually not destroyed, but their activity decreases to almost zero.

The dependence of enzyme activity on the pH of the medium (fig. 14).

Fig. 14. Dependence of the enzymatic reaction velocity on pH.

There is an optimum pH of the medium for each enzyme in which it shows maximum activity. pH optimum of the enzyme lies within a narrow zone of hydrogen ion concentration. It corresponds to physiological values of pH 6.0-8.0 which developed in the course of evolution. Exceptions are pepsin - 1.5-2.5; arginase - 9.5-10.

According to modern concepts, the impact of changes in pH on the enzyme molecule is the impact on the tertiary structure of proteins.

Enzyme specificity.

The high specificity of enzyme action is due to the conformational and electrostatic complementarity between the substrate and enzyme molecules and the unique structural organization of the active site.

The absolute specificity is the ability of the enzyme to catalyze a single reaction. Such enzymes are urease, arginase. Urease catalyzes the hydrolysis of urea to NH₃ and CO₂.

Relative (group) specificity is the ability of the enzyme to catalyze a group of reactions of a certain type. Examples are peptidase, hydrolyzing peptide bonds in proteins and peptides.

Stereospecificity is the ability of enzyme to catalyze the conversion of only one spatial isomer. The enzyme fumarase catalyzes the conversion only of trans-isomer of fumarate and has no effect on the cis-isomer maleic acid.

The high specificity of the enzyme plays an important role in the regulation of metabolism, providing a high speed only to certain chemical reactions of all possible conversions.

 

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