Enzyme rate-determining step

In the normal process ( ), step (J) occurs very rapidly and step (/) is the rate-determining step, whereas in the inhibition process (B), step (3) occurs very slowly, generally over a matter of days, so that it is rate determining. Thus it has been demonstrated with AChE that insecticides, eg, tetraethyl pyrophosphate and mevinphos, engage in first-order reactions with the enzyme the inhibited enzyme is a relatively stable phosphorylated compound containing one mole of phosphoms per mole of enzyme and as a result of the reaction, an equimolar quantity of alcohoHc or acidic product HX is hberated. 

The reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F) has been analyzed extensively14 26-30 and a kinetic scheme for E. Coli DHFR was proposed in which the steady-state kinetic parameters as well as the full time course kinetics under a variety of substrate concentrations and pHs were determined. From these studies, the pKa of Asp27 is 6.5 in the ternary complex between the enzyme, the cofactor NADPH and the substrate dihydrofolate. The second observation is that, contrary to earlier results,27 the rate determining step involves dissociation of the product from the enzyme, rather than hydride ion transfer from the cofactor to the substrate. 

Most catalytic cycles are characterized by the fact that, prior to the rate-determining step [18], intermediates are coupled by equilibria in the catalytic cycle. For that reason Michaelis-Menten kinetics, which originally were published in the field of enzyme catalysis at the start of the last century, are of fundamental importance for homogeneous catalysis. As shown in the reaction sequence of Scheme 10.1, the active catalyst first reacts with the substrate in a pre-equilibrium to give the catalyst-substrate complex [20]. In the rate-determining step, this complex finally reacts to form the product, releasing the catalyst... 

In the monomolecular layer systems described so far, diffusion of the cosubstrate through the film is not a rate-limiting factor. This is true in the case of a free-moving cosubstrate, but also, at least at low scan rates, with cosubstrates attached to the structure. When several layers are coated on the electrode, diffusion of the cosubstrate may become rate limiting even if it is not attached to the structure. The diffusion rate of the two cosubstrate forms increases with its concentration. One may thus expect that the enzymatic reaction, rather than diffusion, tends to be the rate-determining step upon raising the cosubstrate concentration and that this situation is reached all the more easily that the number of layers is small. Under such conditions, the separation of the cyclic voltammetric current in two independent contributions [equation (5.29)] is still valid. icat is thus proportional to the total amount of enzyme contained in the film per unit surface area and therefore to the number, N, of monomolecular layers deposited on the electrode ... 

Although UGTs catalyze only glucuronic acid conjugation, CYPs catalyze a variety of oxidative reactions. Oxidative biotransformations include aromatic and side chain hydroxylation, N-, O-, S-dealkylation, N-oxidation, sulfoxidation, N-hydroxylation, deamination, dehalogenation and desulfation. The majority of these reactions require the formation of radical species this is usually the rate-determining step for the reactivity process [28]. Hence, reactivity contributions are computed for CYPs, but a different computation is performed with the UGT enzyme (as described in Section 12.4.2). 

Specificity constant Defined as kcJKm. It is a pseudo-second-order rate constant which, in theory, would be the actual rate constant if formation of the enzyme-substrate complex were the rate-determining step. 

In many sequential processes, the overall rate of a pathway is determined by the rate of the slowest individual reaction. This is called the rate determining step (RDS) or rate limiting step (RLS).Thus if the rate of an early enzyme catalysed reaction is regulated (increased or decreased) in response to physiological conditions, then the overall rate of the pathway and substrate utilization will be subject to control. 

Figure 11.1 illustrates the behavior of Equation 11.6. By the assumption of rapid equilibrium the rate determining step is the unimolecular decomposition. At high substrate composition [S] KM and the rate becomes zero-order in substrate, v = Vmax = k3 [E0], the rate depends only on the initial enzyme concentration, and is at its maximum. We are dealing with saturation kinetics. The most convenient way to test mechanism is to invert Equation 11.6... 

Equations 2.26 and 2.27 carmot be solved analytically except for a series of limiting cases considered by Bartlett and Pratt [147,192]. Since fine control of film thickness and organization can be achieved with LbL self-assembled enzyme polyelectrolyte multilayers, these different cases of the kinetic case-diagram for amperometric enzyme electrodes could be tested [147]. For the enzyme multilayer with entrapped mediator in the mediator-limited kinetics (enzyme-mediator reaction rate-determining step), two kinetic cases deserve consideration in this system in both cases I and II, there is no substrate dependence since the kinetics are mediator limited and the current is potential dependent, since the mediator concentration is potential dependent. Since diffusion is fast as compared to enzyme kinetics, mediator and substrate are both approximately at their bulk concentrations throughout the film in case I. The current is first order in both mediator and enzyme concentration and k, the enzyme reoxidation rate. It increases linearly with film thickness since there is no... 

Finally, yet another issue enters into the interpretation of nonlinear Arrhenius plots of enzyme-catalyzed reactions. As is seen in the examples above, one typically plots In y ax (or. In kcat) versus the reciprocal absolute temperature. This protocol is certainly valid for rapid equilibrium enzymes whose rate-determining step does not change throughout the temperature range studied (and, in addition, remains rapid equilibrium throughout this range). However, for steady-state enzymes, other factors can influence the interpretation of the nonlinear data. For example, for an ordered two-substrate, two-product reaction, kcat is equal to kskjl ks + k ) in which ks and k are the off-rate constants for the two products. If these two rate constants have a different temperature dependency (e.g., ks > ky at one temperature but not at another temperature), then a nonlinear Arrhenius plot may result. See Arrhenius Equation Owl Transition-State Theory van t Hoff Relationship... 

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