Proteases mechanistic groups

TABLE 12.10 Examples of proteases classified into mechanistic groups... 

The elucidation of the X-ray structure of chymotrypsin (Ref. 1) and in a later stage of subtilisin (Ref. 2) revealed an active site with three crucial groups (Fig. 7.1)-the active serine, a neighboring histidine, and a buried aspartic acid. These three residues are frequently called the catalytic triad, and are designated here as Aspc Hisc Serc (where c indicates a catalytic residue). The identification of the location of the active-site groups and intense biochemical studies led to several mechanistic proposals for the action of serine proteases (see, for example, Refs. 1 and 2). However, it appears that without some way of translating the structural information to reaction-potential surfaces it is hard to discriminate between different alternative mechanisms. Thus it is instructive to use the procedure introduced in previous chapters and to examine the feasibility of different... 

B Elimination has been used to show differences among the disulfide bonds in various proteins including the ovomucoids (54). The e-elimination reaction has also been used to replace the hydroxyl group of the essential serine residue of subtilisin with a sulfhydryl group (55). The thiolsubtilisin had a small fraction of the activity of subtilisin but it has been quite useful in mechanistic studies of the serine and sulfhydryl proteases. 

A final group of covalent small-molecule inhibitors of proteases are mechanism-based inhibitors. These inhibitors are enzyme-activated irreversible inhibitors, and they involve a two-hif mechanism that completely inhibits the protease. Some isocoumarins and -lactam derivatives have been shown to be mechanistic inhibitors of serine proteases. A classic example is the inhibition of elastase by several cephalosporin derivatives developed at Merck (Fig. 8). The catalytic serine attacks and opens the -lactam ring of the cephalosporin, which through various isomerization steps, allows for a Michael addition to the active site histidine and the formation of a stable enzyme-inhibitor complex (34). These mechanism-based inhibitors require an initial acylation event to take place before the irreversible inhibitory event. In this way, these small molecules have an analogous mechanism of inhibition to the naturally occurring serpins and a-2-macroglobin, which also act as suicide substrates. 

Serine hydrolases are enzymes that play a key role in diverse physiological systems. They all use a serine side-chain hydroxy group as a nucleophile in their enzymatic reaction. In contrast to the serine proteases of the trypsin/elastase family discussed above, the two esterases discussed here belong to a different mechanistic class that shares no sequence homology or structural similarity. Lipases digest nutritional fat triglycerides and acetylcholinesterase degrades the synaptic neurotransmitter, acetylcholine. 

The thiol enzyme for which the most detailed mechanistic formulations have been proposed is papain . In this enzyme a cysteine thiol group appears to function in the same manner as the serine hydroxyl of other proteases and esterases. In the hydrolysis of proteins by this plant protease there is an intermediate formation of an acyl thiol, which is subsequently cleaved by water. 

Deslongchamps kindly provided us with his own view of the mechanistic path by which a-chymotrypsin and other serine proteases can hydrolyze secondary amides by stereoelectronic control (Fig. 4.7). Petkov et al. (119) also arrived at a similar proposal by studying the influence of the leaving group on the reactivity of specific anilides in a-chymotrypsin-catalyzed hydrolysis. Furthermore, the stereoelectronic control theory has been applied to the mode of action of ribonuclease A, staphylococcal nuclease and lysozyme (120). 

The two examples that have been chosen are members of the aspartate and serine protease families of enzymes for which more crystal structures are available than for any other homologous group of enzymes. Additionally, representatives of both families have been widely studied mechanistically so, for both reasons, inhibitor design should be relatively straightforward. 

AChE can be classified in several ways. Mechanistically, it is a serine hydrolase. Its catalytic site contains a catalytic triad—serine, histidine and an acidic residue— as do the catalytic sites of the serine proteases (such as trypsin), several blood clotting factors, and others. However, the acidic group in AChE is a glutamate, whereas in most other cases, it is an aspartate residue. 

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