Protein 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... 

These inactivators typically have negligible reactivity toward cellular nucleophiles, in contrast to the classic affinity labels and the activated (escaped) form of suicide substrates (I ). However, all classes of irreversible inactivators - even in the ideal case of covalently labeling only their target enzymes - suffer from the possibility of eliciting an undesired immune response against the inactivator-derivatized protein following protein denaturation and degradation.1171... 

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. 

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... 

These are metabolites that bind primarily to the parent enzyme. This category includes substrates that form enzyme-bound intermediates that react with the active site of the enzyme. Such chemicals are known as suicide substrates. A number of compounds are known to react in this manner with CYP, and such compounds are often used experimentally as CYP inhibitors (see the discussion of piperonyl butoxide, Section 7.2.2). Other compounds, although not true suicide substrates, produce reactive metabolites that bind primarily to the activating enzyme or adjacent proteins altering the function of the protein. 

Vessieres A, Top S, Vaillant C, Osella D, Momon JP, Jaouen G (1992) Estradiol modified by metal carbonyl clusters as suicide substrates for the study of receptor proteins application to the estradiol receptor. Angew Chem Int Ed Engl 31 753-755... 

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]. 

Sometimes called suicide substrates, several protein inhibitors of proteases require proteolytic activity of the enzymes they inhibit, which leads to either covalent modification of the enzyme or releases charged groups that inhibit the catalytic machinery. In either case, this sort of activity-dependent inhibition is powerful and fundamentally different than the competitive mechanisms outlined above the inhibitor acts as a substrate and then uses the enzymes catalytic machinery to trap and then inhibit the enzyme. 

Tetracycline targets protein synthesis in Plasmodium via a similar mechanism to that seen in bacteria inhibition of chain elongation and peptide bond formation. Eflornithine interferes with the metabolism of the amino acid ornithine in T. brucei gambiense by acting as a suicide substrate for the enzyme ornithine decarboxylase. 

This approach was followed concurrendy with that of steroids as part of platinum coordination complexes for which the principal target was found to be DNA. Despite many structural modifications, the results were not spectacular [104-106]. Very often these Pt hybrids or their associated SERMs provided no synergy [107], as far as new, useftil properties were concerned. On the other hand, with covalent organometallic complexes, a number of pioneering results made it possible to study their recognition for specific nuclear receptors, and to develop novel affinity markers or suicide substrates. These preferentially targeted key proteins in specific biological functions to the detriment of DNA. 

When reactive metabolites are formed by metabolic activation, some of them can escape from the active site and bind to external protein residues or be trapped by reduced glutathione (GSH) or other nucleophiles. The remaining molecules that are not released from the active site will cause the suicide inhibition [7]. The ratio of the number of reactive molecules remaining in the active site and those escaping is a measure of the reactivity of the intermediates formed. The addition of scavengers or GSH to the incubation mixture does not affect and cannot prevent the CYP mechanism-based inhibition. However, GSH can reduce the extent of the nonspecific covalent binding to proteins by those reactive molecules that escape from the active site. In contrast, addition of substrates or inhibitors that compete for the same catalytic center usually results in reduction of the extent of inhibition. 

McNamara RK, Hyde TM, Kleinman JE, et al Expression of the myristoylated alanine-rich C kinase substrate [MARCKS) and MARCKS-related protein [MRP) in the hippocampus and prefrontal cortex of suicide victims. J Chn Psychiatry 60 21-26, 1999... 

However, when an irreversible inhibitor binds to the enzyme, it cannot be displaced by the substrate and thus binds irreversibly (Fig. 2—51). The irreversible type of enzyme inhibitor is sometimes called a suicide inhibitor because it covalently and irreversibly binds to the enzyme protein, permanently inhibiting it and therefore essentially killing the enzyme by making it nonfunctional forever (Fig. 2—51). Enzyme activity in this case is only restored when new enzyme molecules are synthesized. 

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