Selection suicide substrate

The starting point for much of the work described in this article is the idea that quinone methides (QMs) are the electrophilic species that are generated from ortho-hydro-xybenzyl halides during the relatively selective modification of tryptophan residues in proteins. Therefore, a series of suicide substrates (a subtype of mechanism-based inhibitors) that produce quinone or quinonimine methides (QIMs) have been designed to inhibit enzymes. The concept of mechanism-based inhibitors was very appealing and has been widely applied. The present review will be focused on the inhibition of mammalian serine proteases and bacterial serine (3-lactamases by suicide inhibitors. These very different classes of enzymes have however an analogous step in their catalytic mechanism, the formation of an acyl-enzyme intermediate. Several studies have examined the possible use of quinone or quinonimine methides as the latent... 

With a 3,3-heterodihalogeno substitution of the (3-lactam ring, a selective interaction of each enantiomer of the chiral azetidinone with the enzyme active site is expected. The enantiomer 3R of the 3F, 3Br derivative indeed has a more favorable kinetic parameter k-JK, than the enantiomer 3S.33 The partition ratio kCA /kt (=k3/k4, Eq. 11.1) for the inactivation is also higher. Therefore, enantiomer 3R is a better suicide substrate for HLE since a lower partition ratio corresponds to abetter suicide substrate.20... 

Methods of this nature are adequate for screening sets of hybridomas but not for selection from much larger libraries of antibodies. So, most recently, selection methods employing suicide substrates (Section 7) (Janda etal., 1997) or DNA amplification methodology (Fenniri et al., 1995) have been brought into the repertoire of techniques for the direct identification of antibodies that can turn over their substrate. However, the tedious screening of hybridomas remains the mainstay of abzyme identification. 

True enough, treatment of PAP with FMPP resulted in a time-dependent inactivation of the enzyme. Competitive inhibitors of PAP protected against inactivation. The authors suggest that FMPP represents a useful basic structure which can be incorporated into the design of more specific phosphatase inhibitors for example, the modified tyrosine 77 could be incorporated into a particular peptide to give a suicide substrate that is selective for a protein phosphatase which preferentially hydrolyses that peptide. 

Fig. 6.1. Summary of methods for selecting phage-displayed enzymes on die basis of catalytic activity (S substrate P product SS suicide substrate Bt biotin SV streptavidin TSA transition state analogue).

As illustrated in Scheme 6.1, once the covalent intermediate is formed, the complex can either follow a normal catalytic cycle or go through a suicide event leading to the irreversible labeling that is necessary for selection. The suicide inhibition efficiency depends on the ratio k /k. This ratio depends on the nature of the suicide substrate and of the enzyme. Therefore, a large excess of suicide substrate as compared to the displayed enzyme is recommended for selection experiments. 

Because the labeling event occurs before the last step of the turnover, and because it even competes with this last step, there is selective pressure for enzymes that feature very low ki rates, that is, enzymes with very low turnover. To overcome this problem, active sites that do not turn over rapidly can be blocked by an initial incubation with a normal substrate prior to labeling with the biotinylated suicide substrate. Hence, special care must be given to the kinetic control of the labeling step. 

The following protocol is described for the selection of phage-displayed serine / -lactamase with a biotinylated penam-sulfone [5] suicide substrate. For other activities, the concentrations of substrate and suicide substrate and the times of reaction should probably be adjusted. 

It can be interesting to increase the selection pressure from one selection round to the next by doubling the number of washes, by decreasing the suicide substrate concentration, or the time of incubation by a factor of 10. This could lead to the selection of the most efficient catalysts. 

So far, only a few groups have reported the application of this technology to libraries. As yet, suicide substrates have been applied for the selection of / -lactamase activity within libraries of mutants containing penicillin-binding proteins [6] and to... 

Suicide substrates have not been applied yet to proteases as extensively as they have to other classes of enzymes such as pyridoxal dependent enzymes (42, 43). The potential high selectivity obtainable with suicide substrates certainly makes pursuit of such inhibitors a worthy goal. 

Another class of cytochrome P-450 inhibitors, compounds with a monosubstituted acetylenic function, are well known for their potential as insecticide synergists (21) and some have already been reported to be active as JH biosynthesis inhibitors as well (19, 22). Ortiz de Montellano and Kunze (23) have shown that many ethynyl substrates cause the destruction of rat hepatic cytochrome P-450, when the prosthetic heme is alkylated during attempted metabolism of the triple bond. Such suicide substrates must bind to the enzyme and be catalytically acceptable thereby offering a potential for selectivity. In fact, selectivity of suicide substrates for particular molecular forms (isozymes) of hepatic... 

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]. 

The selection techniques described above are indirect as they rely on inhibitors. They have important limitations. Transition-state analogues are frequently unable to mimic adequately the essential features of true transition states. For many enzymatic activities, there are simply no transition-state analogues or suicide substrates. 

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