Food Chemistry

Module 6. Food enzymes

Lesson 16
UTILIZATION IN FOOD INDUSTRY AND EFFECT OF INHIBITORS, pH AND TEMPERATURE

16.1 Introduction

A number of factors influence the rate of enzyme catalyzed reactions. The most important factors are Substrate concentration, Enzyme concentration, Temperature, pH, Specific activators, inhibitors. They are discussed below.

16.2 Substrate Concentration

For a given amount of enzyme under standard conditions, the initial reaction velocity varies with an increase of substrate concentration. At a low substrate concentration, the initial reaction velocity is nearly proportional to the substrate concentration (and the reaction is thus approximately first order with respect to the substrate). However, as the substrate concentration is increased, the initial rate falls off and is no longer approximately proportional to the substrate concentration (in this zone, the reaction is mixed order). With a a further increase in the substrate concentration, the reaction rate becomes essentially independent of substrate concentration and approaches a constant rate (in this range of substrate concentration the reaction is essentially zero order with respect to the substrate) and the enzyme is said to be saturated with substrate.

16.2.1 Michaelis-Menten Constant (K_m)

It is an equilibrium constant and is a measure of the affinity of an enzyme for its substrate. The more strongly an enzyme interacts with its substrate, the greater will be the proportion of the enzyme which is combined with substrate as ES, the lower the concentration of free enzyme, E and lower the value for K_m.

[E] + [S] « [ES] ® [E] + P

K_m = [E] [S] / [ES]

K_m = [S], when v₀ = ½ V_max

16.2.2 Enzyme Concentration

For any enzyme, assuming the correct temperature and length of reaction time relationship, a medium at the optimum pH, and a constant substrate concentration, the curve shown in the Fig. 16.2 is valid. If an excess of substrate is present, doubling the enzyme concentration usually doubles the rate of formation of end products. This usually applies at the start of the reaction, for the end products of the reaction often have an inhibitory effect on the enzyme, and decrease its efficiency. As the concentration of enzyme is increase, however, a point could (theoretically) be reached where the substrate (concentration held constant) is saturated with enzymes. If this point could be reached, further increases in enzyme concentration would have no influence on the rate of formation of end products.

16.2.3 Temperature

A curve of the type shown in is usually obtained if enzyme activity is related to variation in temperature. The rate of enzyme-catalyzed reaction at 0°C is close to zero. As the temperature is raised the reaction rate increases until a maximum is reached. At still higher temperature the rate decreases very rapidly back toward zero. The temperature at which the maximum rate is observed is termed the optimum temperature. As the temperature increases, enzyme activity increases such that the rate of most enzymatic reactions approximately doubles for each 10°C rise in temperature, and is usually expressed as the temperature coefficient Q₁₀. The great majority of enzymes show optimal activity within the 30-40^oC temperature range. At about 50^oC, the enzyme becomes inactivated due to the denaturation of the apoenzyme, which results in the unfolding of the molecule and consequent loss of specificity.

The thermolability of enzymes is exploited to a high degree in the food industry. Pasteurization of milk involves exposure of milk to 63^oC for 30 minutes. This treatment is sufficient to kill pathogenic bacteria such as Mycobacterium tuberculosis, and inactivates many enzymes. Effectiveness of pasteurization is determined by the absence of alkaline phosphatase activity. Blanching of fruits and vegetables is an essential pretreatment for fruits and vegetables for canning, freezing, and dehydration. This treatment is normally sufficient to inactivate all enzymes present. The effectiveness of blanching procedure can be determined by the absence of peroxidase activity.

16.2.4 pH effect

Enzymes are very sensitive to changes in the pH of their environment due to their proteinaceous nature. For every enzyme there is an optimum pH, which often lies within the range from 4.5 to 8.0, however, some few are most active in very acidic media, others in quite alkaline solutions. If enzyme activity is related to pH, the type curve shown in Fig. is obtained. Maximum activity is usually observed at or near their isoelectric point. Low catalytic activities are usually found in quite acidic or basic solutions. These effects are due in major degree to the gross denaturation of enzyme protein as well as change in the degree of ionization of functional groups of the enzyme involved in the active centre. Thus a pH change brings about conformation changes in the protein structure, thus altering the active site of the enzyme for its steric fit with the substrate. If enzyme has more than one possible substrate, then the pH optimum can differ from each substrate. (Fig. 16.4)

16.2.5 Specific activators

Many kinases require Mg⁺² ions, carbonic anhydrase requires, zinc, ascorbic oxidase requires copper, salivary amylase requires chloride for their full activities because they form co-ordination compounds and act as bridges between substrate and enzyme (proenzyme activity & coenzymes).

16.2.6 Inhibitors

Reversible Inhibitors : As the term implies the type of inhibition involves equilibrium between the enzyme and the inhibitors, the equilibrium constant (K_i) being a measure of the affinity of the inhibitor for the enzyme. Three distinct types of reversible inhibitors are known. 1. competitive, 2. noncompetitive, 3. uncompetitive.

a) Competitive: Compounds that may or may not be structurally related to the natural substrate combine reversibly with the enzyme at or near the active site. The inhibitor and the substrate therefore compete for the same site according to the above reaction. Succinic acid is the substrate of succinic acid dehydrogenase but its competitive inhibitors are malonic acid, oxalic acid, glutaric and phenyl propionic acid.

Note: Different Km values, No shift in V_max

b) Noncompetitive: Compounds that reversibly bind with either the enzyme of the enzyme-substrate complex are designated as non competitive inhibitors. This type of inhibition is not completely reversed by high substrate concentration since the closed sequence will occur regardless of the substrate concentration. Since the inhibitors binding site is not identical to nor does it modify the active site directly the K_m is not altered.\

c) Uncompetitive Inhibition: Compounds that combine only with the ES complex but not with the free enzyme are called uncompetitive inhibitors. The inhibition is not overcome by high substrate concentrations, interestingly the K_m’ value is consistently smaller than the K_m value of the uninhibited reaction, which implies the S is more effectively bound to the enzyme in the presence of the inhibitor. Uncompetitive inhibition is always a component of noncompetitive inhibition since in both cases EIS is formed.

16.3 Irreversible Inhibitors

Forms a covalent bond with a specific function, usually an amino acid residue, which may, in some manner, be associated with the catalytic activity of the enzyme. In addition, there are many examples of enzyme inhibitors which covalently bind not at the active site, but physically block the active site. The inhibitor cannot be released by dilution or dialysis, kinetically, the concentration and hence the velocity of active enzyme is lowered in proportion to the concentration of the inhibitor and thus the effect is that of noncompetitive inhibition.