Question

1.) Enzyme activity: energy of activation, free energy, how enzymes effect products

2.) Factors affecting activity: concentration of substrate and enzymes, pH, Temperature

Answer 1

Answer

1. Enzymes are biological catalysts. A catalyst is an agent affecting the velocity of a chemical reaction without appearing among the final products of the reaction. Unlike general catalysts, enzymes functioning in the living system have to operate at a temperature of approximately 37°C and pH close to neutrality. Yet, the chemical versatility and ability of enzymes are superior to those of chemical catalysts by a factor of 10⁵ to 10⁸.

Enzymes are distributed among the cells and tissues of plants and animals. Some cellular enzymes are present in solution in the cytosol, while most exist bound to the subcellular organelles. Some subcellular enzymes are characteristic of the organelle, while others occur at different subcellular and tissue locations, catalyzing the same reaction. Some terms often encountered in the study of enzymes need definition. Enzymes are proteins with catalytic properties due to their power of specific activation. It is the protein nature of enzymes that is responsible for their characteristic behaviour and properties. Many enzymes (but not all) depend on the co-operation of non-protein substances called cofactors for their activity. The complete active enzyme, consisting of both the protein and the cofactor, is called the holoenzyme. The protein component without its cofactor is termed apoenzyme. The substance on which the enzyme acts is termed the substrate. Isoenzymes or isozymes are multiple forms of an enzyme occurring in the same species. They catalyze the same reaction and arise from genetically determined differences in the primary structure of the enzyme protein. The cofactors are of two categories—the inorganic cofactors which include simple inorganic ions, such as Zn2+, Mg2+. Mn2+. Fe2+, Cu2+, K+, Na+, etc., and the organic cofactors consisting of about a dozen substances of diverse structure. The organic cofactors are usually called coenzymes.

2. Factors Affecting the Rate of Enzyme Catalyzed Reactions

A number of factors influence the rate of enzyme catalyzed reactions. The most important factors are enzyme concentration, substrate concentration, temperature, pH, electrolytes and the presence of inhibitors. The study of the effect of these factors on the rate of enzyme action is referred to as kinetic studies.

Effect of Enzyme Concentration

For any enzyme, if the temperature, pH and substrate concentration (saturating level) are held constant and if the enzyme concentration [E] is varied, plots of initial velocity (Vo) versus the enzyme concentration are linear . In enzymic reactions, the rate of reaction decreases with time. To minimize errors due to this factor, in enzyme kinetic studies, only initial velocity measurements are made. The straight line relationship between enzyme concentration and initial velocity indicates that the reaction rate is proportional to enzyme concentration

Effect of Substrate Concentration

If, in the above case, the enzyme concentration is kept constant and the substrate concentration is varied, the velocity versus substrate concentration plots are hyperbolic . This shows that the reaction velocity is proportional to substrate concentration only at low concentrations of the substrate and is independent of it at high concentration. To explain this, it is proposed that the enzyme (E) reversibly combines with the substrate (S) to form an intermediate complex of enzyme and substrate (]fS) which then decomposes to yield the product (P) and the free enzyme in its original form. From the concept of ES formation, it follows that at low substrate concentration, not all enzyme molecules are saturated with the substrate and as the substrate concentration increases the enzyme gets saturated with respect to the substrate. Once all the enzyme is in the form of (ES) any further increase in substrate concentration will not increase the rate of reaction, as there will be an equilibrium between [E], [S] and [ES] (or more appropriately as [ES] reaches a steady state).

Effect of Temperature.

Enzyme activity proceeds very slowly at low temperatures. As the temperature increases, the rate of the enzyme-catalyzed reaction increases, as is true of chemical reactions in general. The reaction rate doubles itself for every 10° rise in temperature. However, since enzymes are proteins, thermal denaturation of the apoenzyme sets in as temperature increases, resulting in inactivation of the enzyme. Thus, the temperature effect is the result of two processes, namely, increase in the reaction rate and increase in the rate of denaturation of the enzyme above a critical temperature. If the enzyme activity versus temperature is plotted, a curve is usually obtained. The temperature of maximum activity is not fixed, i.e., there is no optimum temperature characteristic of an enzyme. The temperature of maximum activity depends on time, when other conditions of experiment are constant.

A great majority of enzymes have a temperature of maximum activity between 30-40°C. Inactivation then sets in and is complete above 50 -55°C. There are, however, a few enzymes such as peroxidases which can withstand higher temperatures. Thermolability of enzymes is of great importance in the food industry. In food preservation it is desirable to prevent or control enzyme activity, as otherwise the food product would be of undesirable flavour, poor appearance, altered texture or lowered nutritive value. By the application of heat to food to about 70°C for a few minutes these undesirable effects can be overcome. For example, pasteurization of milk involves exposure of milk to 63°C for 30 minutes. This treatment is sufficient to inactivate the enzymes (or destroy the organisms containing the enzymes), thus retarding milk spoilage. Similarly, blanching of fruits and vegetables by subjecting them to boiling water or live steam for a short neriod inactivates all the enzymes present in them. Thus, the activity of enzymes such as phenolase, lipoxygenase, chlorophyllase and ascorbic acid oxidase are destroyed, and the deterioration of fruits and vegetables during storage is prevented. The effect of low temperature on enzymes is important from the point of view of food preservation. Many enzymes exhibit significant activity in partially frozen systems. At temperatures from 0 to -10°C, enzyme activity can increase or decrease, depending on the enzyme and the system. A further decrease in temperature results in decreased activity.

Effect of pH

The activity of an enzyme depends on the pH of the reaction medium. All enzymes are active only within a narrow pH range and for every enzyme there is a pH of maximum activity known as optimum pH. Most enzymes are active in the pH range of 4.5 to 8.0. There are exceptions: pepsin has a pH optimum of 1.8 and arginase around 10. The pH activity curve is generally bell-shaped. The effect of pH on the enzyme molecule is the alteration of the degree of ionization of the functional groups in the active site of the enzyme, normally required to combine with the substrate and to bring about catalysis. At one ionic condition, the functional groups of the enzyme are most active and thus the enzyme has an optimum pH. At extremes of acidity and alkalinity, denaturation of the protein takes place with a concomitant loss of enzyme activity