Michaelis constant , variation with

These values are then plotted against 1/C . The slope of this linear secondary plot provides k, while the intercept gives the value of I /k 2 + 1/ 2 2- It follows that the two rate constants k 2 and k22 may not be derived separately from this type of experiment. The same is, of course, true for the two Michaelis constants. One has to know the value of one of them independently, or at least know that one is much larger than the other. Dealing with redox enzymes, the variations of the intercept in a series of cosubstrates of increasing reducing power may be used to solve the problem. Indeed, if for the most reducing cosubstrates, the intercept becomes independent of the cosubstrate, one is entitled to conclude that it represents the value of l/k t2. The procedure is illustrated with an experimental example in the next section. 

In the case of carboxylesterase-catalyzed hydrolysis (Table 8.1), the Michaelis constant consistently indicated relatively low affinity for the enzyme, with a variation between substrates of one order of magnitude. Even less variation was seen in the maximal velocity of the reaction. It is interesting to note that, for the four compounds where comparisons are possible, a direct relationship exists between the rate of hydrolysis in plasma and the Vmax of carboxylesterase hydrolysis, suggesting comparable catalytic mechanisms. 

Ionization Constants Determined from Variation of Reaction Velocity and Michaelis Constant with pH... 

Based on in vitro studies of hepatic microsomal-catalysed oxidative reactions, using liver specimens from a variety of animal species including man, and marker substrates for the various reactions, it can be concluded that neither the measured levels of cytochrome P450 and cytochrome nor the activity of NADPH-cytochrome c reductase accounts for species variations in the capacity of oxidative reactions (McManus and Ilett, 1976 Dalvi et al., 1987 Souhaili-El-Amri et al., 1986). However, species variations could be attributed to differences in values of the kinetic parameters (Michaelis constant, Km, and the maximum reaction velocity, Vmax) associated with individual reactions. 

The complete analysis of the primary and the secondary double reciprocal plots is often not necessary to identify the mechanism the mechanism can frequently be determined from the variation of apparent Michaelis constants and maximal rates with the concentration of the fixed substrate (Cleland, 1970, 1977). 

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... 

The Michaelis constants of the two substrates also show striking variations with pH (Fig. 19). These values of Km and have been combined with the dissociation constants of the substrates to permit calculation of pH-independent values for V— and Km- These values apply to a hypothetical enzyme in its active ionic form reacting with the divalent ions of fumarate and malate. The general description of the hypothesis is shown in (III). 

It is also interesting to examine how the plateau current varies with the substrate concentration. For simplicity, we assume that the substrate and cosubstrate concentrations are small enough as compared to the Michaelis-Menten constants for saturation effects to be negligible for both reactions. Then, as illustrated in Figure 5.2, the variations of the plateau current are given by... 

It is apparent that O Eq. 5 is a variation of the MichaeUs-Menten equation. The inhibitor data shown in O Figure 4-7 can instead be fitted to O Eq. 5, holding Km (and Vjnax) constant to their control values (5 pM and 20 nmol/min/mg, respectively). The curve obtained is identical to that fitted with the Michaelis-Menten equation (O Figure 4-7), but nonlinear regression now yields the information that K, of the inhibitor equals 40% of the concentration at which it was included in the assay to obtain the best-fit curve. In other words, if the concentration of inhibitor present in the experiment shown in O Figure 4-7 was 25 pM, the Ki for the inhibitor is 10 pM. 

Multiple substrate mechanisms follow Michaelis-Menten kinetics. Experiments are performed with constant concentrations of the enzyme and one substrate with variation of the second substrate concentration ([S2]). (Note that the second substrate concentration [S2] is not the same as a deceptively similar term, the square of the substrate concentration [S]2.) Plotting V against [S2] gives a hyperbolic curve and allows determination of Km for the second substrate. The Km values for all substrates may be found in a similar fashion. 

If the concentration of B is held constant, the variation of v with [A] is of the Michaelis-Menten form. This may be shown for the case in which we have an excess of B. Thus, if [B] is sufficiently large, we may neglect the first two terms in the denominator of (10,26), and the result is... 

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