Suicide substrates, selection with

The irreversible inhibition of enzymes by suicide substrates occurs as a consequence of activation steps in which the target enzymes transform these substrates into inhibitors using their normal mechanism. 

This strategy has been applied to select catalytic antibodies from phage-displayed libraries. Two catalytic single-chain antibodies catalyzing the hydrolysis of ampicillin with rate accelerations kcit/kunc lt of 5200 and 320 (kcat = 0.29 and 0.018min-1) were isolated from combinatorial libraries prepared from mice immunized with penam sulfone conjugates and selected with a biotinylated penam sulfone [61]. 

Antibodies possessing glycosidase activity have been induced by immunization of mice with conjugates of the transition-state analogue 21 (Fig. 5.10A). A combinatorial phage-Fab library was then constructed by PCR amplification of the heavy and light chains. Selection with a BSA-conjugated suicide inhibitor 22 (Fig. 5.10B) was then 

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. 

In order to avoid these limitations, efforts have been made to find ways of coupling substrate turnover directly to a selection process. Two groups have devised a method that involves the attachment of a substrate to a phage-enzyme in a way that allows its intraphage interaction with the enzyme. Phage-displaying active enzymes are able to convert the substrate into product. They can be captured from mixtures with product specific reagents or antibodies. 

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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]. 

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... 

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... 

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. 

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. 

During the past three decades, besides the rational design of hundreds of molecules that have been synthesized and tested as suicide substrates. It also has come to light that nature Itself has known about this mechanistic mode of enzyme Inhibition and provided us with several extremely potent mechanism-based suicide Inactivators. Below are a few selected examples to demonstrate the mode of action of these Inhibitors. 

Suicide Substrates. Much like affinity labels, suicide inhibitors first form a reversible complex with the target enzyme due to the structural similarity between inhibitor and substrate. In a subsequent time dependent step, an irreversible complex (usually covalent) is formed with an appropriately positioned amino acid side chain. Unlike the affinity label, suicide substrates (65) are not inherently reactive and must undergo activation by the target enzyme before the irreversible complex is formed. Therefore, these inhibitors are generally more selective than affinity labels. Since the enzyine catalyzes its own inactivation, these inhibitors are also known as kcat inhibitors (66), enzyme-activated, irreversible inhibitors (54) and Trojan horse reagents (6. ... 

Inhibition can be reversible when it simply complexes at the active site preventing further catalysis. The active enzyme under these conditions can be recovered by dialysis. Another form of inhibition is the irreversible type where the active enzyme cannot be recovered by dialysis. A variant of this type of inhibition is suicide inhibition a substrate of the enzyme reacts at the active site to yield an irreversible inhibitor which then reacts directly with groups at the active site [18]. A technique, in situ click chemistry , is related to that of suicide inhibition and involves click chemistry components which complex at the active site of an enzyme and combine to form femtomolar inhibitors. The technique can be used to synthesise inhibitors or by selection from a library of click chemistry components to search structure space of the inhibitor for the drug target [ 19]. 

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