Enzyme kinetics variation study

This is exactly what one would expect for binding of deprotonated enzyme to protonated inhibitor. The pH of maximal inhibition ( 7) is well removed from the pH optimum of catalysis (<5). However, as with all pH variation studies (see the discussion of kinetic equivalence, Section 3.7.4), information is provided on the composition of the complex, not its structure. If we set B = AK j Ki, then eqn. (5.28) holds, giving the appearance of the binding of protonated enzyme to deprotonated inhibitor. pH variation studies cannot by themselves make the distinction between the two modes of binding. 

This study has important lessons for enzyme kinetic analysis. The use of pH variation and examination of isotope elfects can be a powerful combination to explore the chemistry of enzyme-catalyzed reactions and to dissect the contributions of individual reaction steps to the net steady-state turnover (27). Examination of the effects of pH on each step of the reaction pathway could resolve the contributions of ionizable groups toward ground-state binding energy and transition-state stabilization. The use of isotope effects by transient-state kinetic methods is more limited than in the steady state due to the errors involved in comparing two rate measurements. In the steady state, the ratio method has allowed isotope effects of less than 1% to be measured accurately (8a, 58). By transient-state kinetics, one would require at least a 10-20% change in rate to demonstrate a convincing difference between two rate measurements in most instances. 

D-glucose-6-phosphate (G6P) to D-glucono-5-lactone-6-phosphate while reducing NADP+ to NADPH. The reaction kinetics of this enzyme in solution as well as in sol-gel media were studied to elucidate differences in reactivity. The Michaelis-Menten kinetics of the enzyme reveal variation in the kinetic parameter in the sol-gel medium as compared to buffered solution. An approximately fourfold increase in die value of Michaelis constant is observed in the aged gel (78.4 M) as compared to solution (21.7 M). The increase is consistent with an overall shift in the equilibrium towards free... 

Some of the results obtained by differential centrifugation showed enzyme distribution between different cell fractions which were difficult to interpret. Enzymes like carbamoyl phosphate synthase or isocitrate dehydrogenase were found both in mitochondria and in the soluble fraction of the cell. This led to detailed kinetic studies with purified enzymes which indicated there might be populations of enzymes with slightly different properties (isozymes) catalyzing similar reactions in different compartments or in different cell types. Variations in kinetic behavior appeared to tailor the enzyme appropriately to the particular compartment or cell where the reaction took place. 

A related assay was later reported which makes use of a different and more convenient indicator (bromothymol blue) 78). However, it is emphasized that all of the assays based on pH change reported so far refer to the use of isolated enzymes. In real applications, supernatants are likely to be used, as in directed evolution studies. In supernatants, however, pH variations may occur. Therefore, an optimized assay was later developed in which supernatants are employed (79). Thus, the pH of the buffer is adjusted to the acidity of the medium, enabling about 4000 samples in kinetic resolution investigations to be screened per day. 

A coating bearing one enzyme (papain) is produced on the surface of a glass pH electrode by the method previously introduced (co-crosslinking). The papain reaction decreases the pH, and the pH-activity variation gives an autocatalytic effect for pH values greater than the optimum under zero-order kinetics for the substrate (benzoyl arginine ethyl ester) the pH inside the membrane is studied as a function of the pH in the bulk solution in which the electrode is immersed. A hysteresis effect is observed and the enzyme reaction rate depends not only on the metabolite concentrations, but also on the history of the system. 

The inhibitor constants, Kj, may be determined by measuring the variation of v with [S] at different concentrations of the inhibitor. Lineweaver—Burk plots can then be used to find K/ from measurements made in the presence, and absence, of inhibitor (see Fig. 2 and Table I). The kinetics of the inhibition of such enzymes as alpha-amylase, hefo-amylase, and phosphorylase have been studied, and inhibitor constants evaluated. 

The effect of temperature on the kinetics of the enzyme-catalyzed reaction is further complicated by changes in the pK s of the ionizable groups at the active center of the enzyme, thus producing additional changes in V and Km- A study of the variation of these pK s with temperature yields information on the heats of ionization of the ionizable group, AHj, by use of the equation AHj = —2.303 RT .dpK/ dT. The value of AHj has been calculated for the ionizing groups of bacterial alp/io-amylase and potato phosphorylase. ... 

The effect of temperature on the kinetics of alpha-amylase action has been studied, and, from the variation of reaction velocity, activation energies have been determined for several enzymes, as shown in Table V. The variation of with temperature has also been studied, and, from such measurements, the heat of formation of the enzyme—substrate complex has been calculated to be —3.4 Kcal. per mole for sorghum alpha-amylase and —7 Kcal. per mole for hog-... 

The use of pH variation and isotope effects in transient kinetics can be illustrated with a recent study on dihydrofolate reductase. Analysis by steady-state methods had indicated an apparent p/fa of 8.5 that was assigned to an active site aspartate residue required to stabilize the protonated state of the substrate (59). In addition, it was shown that there was an isotope effect on substitution of NADPD (the deuterated analog) for NADPH at high pH but not at low pH, below the apparent p/fa This somewhat puzzling finding was explained by transient-state kinetic analysis. Hydride transfer, the chemical reaction converting enzyme-bound NADPH and dihydrofolate to NAD+ and tetrahydrofolate, was shown to occur at a rate of approximately 1000 sec at low pH. The rate of reaction decreased with increasing pH with a of 6.5, a value more in line with expectations for an active site aspartate residue. As shown in Fig. 14, there was a threefold reduction in the rate of the chemical reaction with NADPD relative to NADPH. Thus direct measurement of the chemical reaction revealed the full isotope effect. 

Kinetic and structural characterization of the known CoADR enzymes shows the occurrence of some variation with regard to their specificity. The S. aureus CoADR, a homodimer of 49kDa subunits, has a of 11 2 pmol 1 and 1.6 0.5 pmol for CoA disulfide and NADPH, respectively. Although an initial study of this enzyme reported ifcat values in the range of 1000 s (which would have had the enzyme operating near the limiting rate constant for a diffusion-controlled enzyme—substrate encounter), " subsequent kinetic analyses have shown the turnover number to be closer to 27 The enzyme shows 17% activity in the presence of... 

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