Enzyme kinetics, which investigates the rates of enzyme-catalyzed reactions as affected by various factors, offers an enormous potential to the study of enzyme reaction mechanisms and functions. Some important factors that affect the rates of enzymatic reactions are enzyme concentration, ligand (substrates, products, inhibitors, and activators) concentrations, solvent (solution, ionic strength, and pH), and temperature. When all these factors are properly analyzed, it is possible to learn a great deal about the nature of enzymes. The kinetic studies of an enzymatic reaction by varying ligand concentrations provide kinetic parameters that are essential for an understanding of the kinetic mechanism of the biochemical reaction. Studying an enzyme's kinetics can reveal the catalytic mechanism of this enzyme, its role in metabolism, how its activity is controlled, and how a drug or an agonist might inhibit the enzyme.
Substrate Concentration Effect
The rate of many chemical reactions increases with the increase of the concentration of substrates. For a single substrate irreversible enzyme reaction, when the substrate concentration increases, the rate of enzyme reaction increases and approaches a maximum value. Based on the saturation of substrate to enzymatic reaction, the theory of “intermediate product” is put forward by German biochemist Leonor Michaelis and Canadian physician Maud Menten: the enzyme (E) binds to the substrate (S) to form an unstable intermediate product or complex (ES), and then produces the product (P) and free enzyme (E). The relationship between reaction rate and substrate concentration was derived by the theory, that is, Michaelis–Menten equation.
When the concentration of substrate in the reaction system is high enough, the enzymatic reaction rate is proportional to the enzyme concentration. The amount of enzyme present in a reaction is measured by the activity it catalyzes. The relationship between activity and concentration is affected by many factors such as temperature, pH, etc. An enzyme assay must be designed so that the observed activity is proportional to the amount of enzyme present in order that the enzyme concentration is the only limiting factor.
Any substance that can reduce the rate of enzymatic reaction, but does not cause denaturation and inactivation of enzymes, is collectively referred to as an enzyme inhibitor. According to the inhibitory effect of inhibitors, they can be divided into two categories: irreversible inhibitors and reversible inhibitors. Irreversible inhibition is caused by the covalent binding of inhibitors with the essential groups of enzymes. Simple methods such as dialysis cannot be used to restore enzyme activity. Reversible inhibition is caused by the non-covalent binding of inhibitors with enzymes. Simple methods such as dialysis can be used to restore enzyme activity. Reversible inhibition includes competitive, uncompetitive and non-competitive inhibition.
Generally speaking, the enzymatic reaction rate increases with the increase of temperature, but when the temperature increases to a certain point, the reaction rate decreases rapidly due to the thermal denaturation of enzyme. The temperature at which the enzymatic reaction rate reaches a maximum is called the optimum temperature of the enzyme. The optimum temperature of the enzyme is related to the experimental conditions, so it is not the characteristic constant of the enzyme.
The concentration of hydrogen ion in the reaction medium also greatly affects the activity of the enzyme. The enzyme often shows the maximum activity within a certain pH range, and the pH, when the enzyme shows the maximum activity is the optimal pH of the enzyme. In the optimum pH range, the enzyme reaction rate is the highest, otherwise the enzyme reaction rate will decrease.
Enzymes do not make reactions take place, they stimulate the rate at which reactions do take place. Any chemical reaction which proceeds in the presence of an enzyme will also proceed in the absence of the enzyme but at a much slower rate. Enzymes catalyze the rate of chemical reactions by lowering the activation energy of the reaction, and they do this in a manner which is highly specific for the reactants of the reaction. It was realized very early in the study of enzyme action that meaningful studies of enzyme action would, of necessity, involve the study of the kinetic behavior of the chemical reaction in the presence of the appropriate enzyme. It is still true that if one understands the kinetic behavior of the enzyme-catalyzed reaction, one also understands much about the mechanism of the enzyme reaction. This requires the investigation of the kinetic behavior of the enzyme reaction under conditions which are defined meticulously. The two most important kinetic properties of an enzyme are how easily the enzyme becomes saturated with a particular substrate, and the maximum rate it can achieve. Knowing these properties suggests what an enzyme might do in the cell and can show how the enzyme will respond to changes in these conditions.
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