Slow binding inhibitors 2- step mechanism

Slow-binding inhibitors operate by one of two mechanisms. Either the inhibitor binds slowly in an initial step, or the initial binding step occurs quickly, followed by a slow rearrangement of the E-I complex. 

Figure 6.2 Effect of preincubation time with inhibitor on the steady state velocity of an enzymatic reaction for a very slow binding inhibitor. (A) Preincubation time dependence of velocity in the presence of a slow binding inhibitor that conforms to the single-step binding mechanism of scheme B of Figure 6.3. (B) Preincubation time dependence of velocity in the presence of a slow binding inhibitor that conforms to the two-step binding mechanism of scheme C of Figure 6.3. Note that in panel B both the initial velocity (y-intercept values) and steady state velocity are affected by the presence of inhibitor in a concentration-dependent fashion.

Slow-Binding Inhibitors. Two different mechanisms have been suggested to rationalize the slow-binding behavior of competitive inhibitors (71, 78, 80). In the one-step mechanism A, the direct binding process of the inhibitor to the enzyme is slow (Equation 17.26) that is, the magnitude of 3[I] is small relative to i[S] and k, the rate constants for the conversionof substrate to product. 

The first mechanism-based inhibitor (11) of 3-deoxy-D-arahino heptulosonate 7-phosphate (DAH7P synthase) has been synthesized in 12 steps from D-arabinose and has been found to be a very slow binding inhibitor against E. coli DAH7P synthase/ ... 

The inhibitor could be displaced from Factor Xa by substrates and, based on steady-state assumptions, the dissociation constant for (19) was found to be 14 pM (87). However, the reaction progress curves indicated a slow-binding process, probably by mechanism B. Stopped-flow fluorescence studies, combined with kinetic analysis, showed that the isomerization step (E. I -I- E. I ) is unusually fast and that the formation of E I is, at least, partially rate limiting. 

Most mechanisms for slow binding inhibition involve one or two kinetic steps during association of the inhibitor with the enzyme.32 In the one step mechanism, a high affinity complex (E-I, apparent inhibition constant K ), is generated directly, without any detectable intermediates. In the two-step process, there is an initial complex (E-I, apparent inhibition constant K ), in equilibrium with uninhibited enzyme, followed by a subsequent tightening to give the final complex (E-I ) with an overall steady state apparent inhibition constant K. ... 

Interestingly, although many transition state analogs bind noncovalently to the target enzyme s active site via a one-step kinetic mechanism (Scheme la) and would therefore be expected to exhibit no time-dependent properties of inhibition, inhibitors with Kj values of < 10 10 M (like coformy-cin) usually have a slow onset of inhibition kobserved < 10 2 s 1 (i.e., an approach to equilibrium inhibition of > 1 min).161 This is merely an assay artifact due to... 

This scheme is analogous to that of the Michaelis-Menten mechanism, and the reaction should thus show saturation kinetics with increasing inhibitor concentration. The kinetics were solved in Chapter 4, equation 4.46. For the simple case of pre-equilibrium binding followed by a slow chemical step, the solution reduces to... 

This strategy has also been applied for the selection of active //-lactamases from a library of mutants also containing penicillin-binding proteins. For this purpose, the protocol had to be modified to circumvent a difficulty of selections with suicide substrates in mechanisms involving a covalent intermediate. If inhibition arises from a covalent intermediate (Y in Scheme 5.2, an acyl-enzyme in the case of serine //-lactamases), enzymes whose rate of release of this intermediate (hydrolysis of the acyl-enzyme) is slow will be efficiently selected as the efficiency of inhibition depends on the ratio of rate constants k4/k3 (Scheme 5.2). To prevent the selection of enzymes with inadequate turnover, a counter-selection step was included in the protocol the library of mutants was incubated with substrate in order to block them as covalent intermediates before adding the biotinylated inhibitor. The library could be enriched from 6 ppm to 25 % active //-lactamases in four rounds of selection [62]. 

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