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Chapter Iii Enzyme Kinetic Studies

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Chapter Iii Enzyme Kinetic Studies CHAPTER III ENZYME KINETIC STUDIES ON BANANA FRUIT PULP AND MUCUNA PRURIENS PPO VELVET BEANS ENZYME KINETICS 3.1 INTRODUCTON TO ENZYME KINETICS : Kinetics, in conjugation with other techniques like x-ray crystallography, provides valuable information on the mechanisam of action of an enzyme. It can give an insight into the role of an enzyme under conditions which exist in the cell and the response of an enzyme to changes in the concentrations of motabolites. Kinetics can help to show how the activity of an enzyme can be controlled, which may provide a valuable pointer to mechanism of regulation under physiological conditions. The charecteristic property and function of enzyme is the catalysis of chemical reactions. Any fundamental study of this catalytic function must be based on quantitative measurements of the rate of the catalysed reaction. From the effect of varying the conditions on the rate, inferences may be made about the mechanism of enzyme action. Hence kinetic studies should be brought into relation with chemical and structural studies on the enzyme in order to get a definite picture of the process. It is well known that biological systems, particularly living cells, are more sensitive to changes of temperature, enzyme concentration, substrate concentration, ionic strength and pH etc. than non-biological chemical reactions and this is due largely to the properties of the enzymes on which these systems depend. A 227 knowledge of enzyme kinetics thus helps in the understanding of many biological phenomena. Without the knowledge of the kinetics of an enzyme it is not possible to solve the problem of how an enzyme works in chemical terms or how it functions in the cell. Thus the kinetic study of the enzyme is necessary for the design of experiments and the interpretation of the results and can be regarded as being an essential foundation to any studies of an enzyme. Enzyme kinetics is that branch of enzymology which deals with the factors affecting the rates of enzyme catalysed reaction. The most important factors are those of enzyme concentration, substrate concentration, pH, ionic strength and temperature. Proper analysis of all these factors allows one to learn a great deal about the nature of the enzyme catalysed reactions. For example, by varying substrate cencentration one can deduce the kinetic mechanism of the reaction. Such studies can establish the kinds of enzyme substrate and enzyme product complexes that can form and in some cases provide evidence for stable covalentely bound intermediates that are undetectable by ordinary chemical analysis. From the kinetic data one can obtain information about the usual intracellular concentration of substrates and products, the physiological direction of the reaction and the mechanism of regulation of enzyme in vivo. pH and temperature studies provide valuable information concerning the identities of amino acid residues at the active site. 228 The studies of effect of various factors on enzyme reaction can help in optimising the reaction conditions for a particular enzyme reaction and hence output of the reaction products can be increased. Kinetics parameters of pure PPO from mucuna pruriens and banana fruit pulp were studied extensively, e.g. substrate concentration, enzyme concentration, pH., temperatureand ionic strength. The effect of various factors mentioned above on the mucuna pruriens and banana pulp polyphenol oxidase (PPO) reactions is described in this chapter. 3.2 Factors influencing enzyme reaction velocity : The factors which determine the form of the progress curves of enzyme reactions are numerous and varied. It is therefore difficult to derive kinetic equations which will represent the curves. In some cases the conditions are sufficiently simple to allow a reasonable close approximation to be made. The best method of investigation is to very one factor at a time, keeping the other conditions constant and to determine the effect on the initial velocity of the enzyme reaction. The chief factors which determine the initial velocity of a particular reaction are enzyme concentration, substrate concentration, pH, ionic strength temperature and the presence of activators or inhibitors. T>Q 3.3 Effect of enzyme concentration : Under given conditions, two molecules of enzyme acting independently in solution will transform twice as much substrate in a given time as one molecule; the velocity should therefore be proportional to the enzyme concentration. y=K[E] This is actually found in the majority of cases and indeed is the basis of nearly all methods of estimation of enzymes, but deviations can occure, and a linear relationship between velocity and enzyme concentration in the range used should be established before any further attempt at kinetics or specific activities are calculated. The linear curve of the enzyme, when velocity is ploted against enzyme concentration, may show an upward or downward curvature at higher concentrations of the enzyme also. Frequently, departures from linearity may be attributed to an artefact of the system or in some cases such behaviour becomes the property of the enzyme itself. Therefore the rate of an enzyme catalysed reaction is directly proportional to the concentration of enzyme provided the pH and temperature parameters of the system are kept constant and the substrate concentration is present in excess. PRESENT STUDIES : To study the effect of the enzyme concentration on the rate of conversion of substrates like tyrosine, 1-dihydroxyphenyl alanine (dopa) and 4 methyl catachol, 230 O CL CL 3 a 01 MSUNfUAlIAUDV CO E ) A UJ 1 oo >• OXIDAS UJ 01 * (SlINH) A1JAI1DV M A UJ > rsi M u DOP m Z o UJ UJ — i£> 4 1 U) * » 1 / to d 01 x (SUNn)AUAUDV 20 60 100 00PA5E ENZYME CONC. IN jug FIG.3.2 :ENZYME CONCENTRATION PROFILE 2 4 6 2 4 6 ( DOPA ) mM ( TYROSINE ) m M FIG. 3 .3 EFFECT OF SUBSTRATE CONCENTRATION ON BANANA PULP PPO the concentration of mucuna pruriens and banana pulp Protein was varied from 30- 150jj.g and 8-40 u.g respectively. The reactions were carried out as described in assay methods( page no* l06-107 \ The dopachrome formed was measured calorimetrically at 460nm and at 265nm for 4-mc oxidase. RESULTS: Tyrosinase and dopa oxidase were isolated in pure form from mucuna yruriens. tyrosinase, dopa oxidase and 4- mc oxidase were isolated in pure form from banana fruit, pulp and kinetic studies were carried out for PPO of both species. The results presented in Table No.3.1 & 3.2 and Fig. (3.1 & 3.2) indicate that under assay conditions, the formation of quinone is directly proportional to the enzyme concentration and holds a linear relationship at least upto 10u.g of protein for tyrosinase, dopa oxidase of mucuna pruriens and for banana pulp tyrosinase, dopa oxidase and catacholase, linear upto 40u.g of protein. The nature of the graphs shows upward curvature. It may be due to the effect of subunits of the enzyme on each other. Hence in every kinetic experimental investigations 10 and 40u.g of the enzyme were used. 231 TABLE NO. 3.1 EFFECT OF ENZYME CONCENTRATION ON MUCUNA PRURIENS PPO ENZYME CONCENTRATION ACTIVITY UNITS (ji moles/min.) (micro grams) TYROSINASE DOPA OXIDASE 2 0.24 0.36 4 0.42 0.68 6 0.52 0.88 8 0.54 1.10 10 0.56 1.20 20 0.48 1.54 40 1.64 TABLE NO.3.2 EFFECT OF ENZYME CONCENTRATION ON BANANA PULP PPO : ENZYME ACTIVITY UNITS (fj. moles/rain.) CONCENTRATION TYROSINASE DOPA OXIDASE 4-MC OXIDASE (MICROGRAMS) 08 0.325 0.275 0.333 16 0.625 0.475 0.583 24 0.800 0.675 0.783 32 1.000 0.875 0.883 40 1.000 1.000 1.000 232 3.4 EFFECT OF SUBSTRATE CONCENTRATION ON PPO ACTIVITY : The vast majority of enzyme catalysed reactions involve more than one substrate. This is true for the hydrolases which are frequently treated as being single substrate reactions since the second substrate (water) is present in such high concentrations that it is usually assumed that the enzyme is saturated with this substrate at all times. Although the single substrate enzyme reaction is something of a rarity in biochemical systems, it nevertheless represents a very useful single model for the development of theories of enzyme kinetics and the majority of the conclusions reached are directly applicable to more complicated enzyme systems. Substrate concentration is one of the most important factors which determine the velocity of enzyme reactions. In most cases when initial velocity (Vo) is plotted against substrate concentration (S) a reaction of the rectangular hyperbola is obtained. The enzyme (E) first forms a (E-S) complex with its substrate (S) which subsequently breaks down giving the free enzyme (E) and the products (P) of the reaction/ 479'480 } K+l E + S • ES K-l ES ( + a second reactant) k + 2 E + P where, k +1, k _x and k+2 represent the velocity constants for the individual reactions if a second substrate is involved, its concentration should be included in k+2. 233 A simple theory involving this type of process was first put forward in 1913 by Michaelis and Menten equation(481). The rate equation for a one substrate enzyme catalysed reaction is - Vmax [SJ V0= Kn, +[S] This M-M equation relates the initial velocity (Vo), the maximum velocity (Vmax) and the initial substrate concentration [S] through the Michaelis-Menten constant [Kl At low substrate concentration, the initial velocity (Vo) in proportional to the substrate concentration, while at high substrate concentration the reaction rate is slowed down to such an extent that it becomes almost constant. Further increase in substrate concentration does not increase the reaction rate. But in certain cases the velocity may fall again at high substrate concentration i.e.
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