Scissile bond

Figure 11.4 Serine proteinases catalyze the hydrolysis of peptide bonds within a polypeptide chain. The bond that is cleaved is called the scissile bond. (Ra) and (Rb)j/ represent polypeptide chains of varying lengths.

Even though these enzymes have no absolute specificity, many of them show a preference for a particular side chain before the scissile bond as seen from the amino end of the polypeptide chain. The preference of chymotrypsin to cleave after large aromatic side chains and of trypsin to cleave after Lys or Arg side chains is exploited when these enzymes are used to produce peptides suitable for amino acid sequence determination and fingerprinting. In each case, the preferred side chain is oriented so as to fit into a pocket of the enzyme called the specificity pocket. 

Cleavage occur s at the scissile bond. Residues in the substrate towards the N-terminus are numbered PI, P2, P3, etc, whereas residues towards the C-terminus are numbered PI, P2, P3 etc. Cleavage occurs between PI and P1. For a peptidase with limited specificity, only the residue in PI or PI is important for specificity. A peptidase with an extended substrate binding site will have a preference for residues in other positions. For example cathepsin L prefers substrates with phenylalanine in P2 and arginine in PI. However, this is a preference only, and cathepsin L cleaves substrates after other amino acids. Caspase-3 has a preference for Asp in both P4 and PI, but it is unusual for substrate specificity to extend much further from the scissile bond. The peptidase with the most extended substrate specificity may be mitochondrial intermediate peptidase that removes an octopeptide targeting signal from the N-terminus of cytoplasmically synthesized proteins that are destined for import into the mitochondrial lumen. 

With this information in hand, initial attempts to generate BACE inhibitors used the peptidic Swedish variant substrate as a starting point and substituted the scissile amide bond with a statine. For example, Sinha et al. (1999) synthesized a P10-P4 1 Swedish variant peptide with a statine moiety in place of the Pl-Pl scissile bond and showed that this peptidomimetic displayed an IC50 of 40 pM for inhibition of BACE. Optimization of this inhibitor was then performed by systematic replacement of amino acid side chains. Replacement of the PE Asp residue by Val reduced the IC50 for BACE inhibition to 30 nM this inhibitor is referred to here as Stat-Val. 

The central feature that defines all DUBs is that they recognize and act at the C-terminus of the ubiquitin or ubiquitin-like domain. All mature ubiquitin and ubiquitin-like proteins have a C-terminal gly-gly motif and DUB cleavage releases leaving groups attached to the carboxyl group of the C-terminal glycine. With the exception of the JAMM metalloproteases, DUB catalysis starts with the nucleophilic attack of the catalytic cysteine on the carbonyl carbon of the scissile bond to... 

Proteinases (Prt), again by definition, are responsible for the cleavage of peptide bonds, usually with a preference for particular types of side chain on the amino acid residues on one or the other or both sides of the scissile bond (Equation (7)). 

A number of other scissile bond replacements have been explored in an attempt to produce inhibitors of the bacterial collagenases, but all have exhibited relatively low potency Table 8.6). These include a compound with an A-methylamide residue in subsite P l (46), two carboxylates (47)-(48), the hydroxamate N-terminal (49) and C-terminal analogues (50)-(51), and one A-carboxyalkyl (52) derivative. 

The selection of inhibitors considered in this section is complete in terms of the scissile bond surrogate functionalities that have been investigated, and representative in terms of residue substitutions, but it is not exhaustive... 

The hydroxamate functionality is an effective scissile bond surrogate in collagenase inhibitors [1,186-203], probably because of its ability to serve as a bidentate ligand for the active site Zn(II). N-terminal tri- and tetrapeptide hydroxamate substrate analogues are only moderately potent inhibitors Table 8.15). An intriguing observation with respect to possible binding modes is that the residues in subsites P and P2 can be replaced with their D-stereoisomer counterparts with essentially no loss of potency, as long as both are replaced with the D-isomers (compare (151), (152) and (153), and (154) vs. (157), in Table 8.15). 

A number of 7V-carboxyalkyl and A-phosphonoalkyl substituted substrate analogue inhibitors have been examined [161,204-208]. These derivatives contain both the acidic carboxylate (or phosphonate) and basic amine functionalities in the vicinity of the scissile bond. Thus, they are capable both of electrostatic interaction with the active site Zn(II) and hydrogen bonding interactions with other active site residues. They are, however, only moderately potent collagenase inhibitors Table 8.18). The stereochemistry at the carbon atom to which the carboxylate moiety is bonded markedly influences the inhibitory potency of these derivatives ((197) vs. (198)). The phosphonate analogues of this class of derivatives have also been evaluated Table 8.18), but are not substantially better inhibitors than the carboxyl-ates. 

Included in this category of compounds are one example of a substrate analogue containing an 7V-ketoalkyl scissile bond surrogate moiety (213), and a number of non-substrate analogue peptides Table 8.19) [176, 209-214]. Some of the latter compounds contain a Cys residue whose side-chain may be interacting with the active site Zn(II) atom. All of these compounds are... 

Scheme 10.5 Peptide encoded combinatorial peptide libraries via enzyme-mediated spatial segregation. P-P. Substrate with a scissile bond between P and P, S. Terminal Residue of the screening structure, C. Terminal Residue of the coding structure.

Serine Proteinases. This group of proteinases is the best known because they are more numerous and better characterized than the other three groups. These proteinases are found in virtually all organisms, indicative of their importance and wide ranging proteolytic capabilities. They demonstrate broad substrate specificities with the sites (amino terminal to the scissile bond) generally being more important in enzyme interaction. 

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