Penicillopepsin transpeptidation

Acid proteases are inactivated by active-site specific reagents, diazoacetylnorleucine ethyl ester and other diazo compounds, and epoxy (p-nitrophenoxy)propane. Covalently labelled aspartic acid peptides have been isolated from pepsin, chymosin (= rennin), and penicillopepsin. The peptides labelled with the diazo compounds have similar sequences and differ from the epoxy (p-nitrophenoxy)pro-pane labelled peptides. These results indicate two aspartic acids at the active site and suggest homology between the enzymes. The latter is confirmed by a comparison of the sequence data. Studies of the action of porcine pepsin and penicillopepsin on some dipeptides with free N-terminal groups show transpeptidation involving a covalent acyl intermediate. It is proposed that there are differences in the mechanism of action of pepsin which are determined by the nature of the substrate. 

Four reviews dealing with the mechanism of action of pepsin have been published in recent years (46, 73, 108, 117). Other recent publications deal with various aspects of this mechanism 118-120). In this section, therefore, the main emphasis will be placed on the significance of studies on hitherto unobserved pepsin- and penicillopepsin-catalyzed transpeptidation reactions, especially as they aflFect the mechanisms proposed by various authors. The question we are concerned with is the role of the two carboxyl groups which are involved in the catalytic action. We shall not further consider the role of other functional groups which have been discussed in the previous section. 

However, since the time these mechanisms were proposed, we have obtained more direct evidence for an acyl intermediate in pepsin- and penicillopepsin-catalyzed reactions from transpeptidation reactions which only proceed via an acyl transfer 129). Some of the experimental evi-... 

This ambiguity was resolved in the experiment shown in Table XL The peptide in this case represents the C-terminal sequence of the B-chain of insulin. The major products were the penta-peptide lacking the N-terminal phenylalanine and free phenylalanine indicating that hydrolysis was the major reaction. In addition, however, there was a significant amount of Phe-Phe. This dipeptide could only come from a transpeptidation involving an acyl transfer. The acyl transfer presumably proceeds via a covalent acyl intermediate. The evidence for this comes from the experiment shown in Table XII where Leu-Tyr-Leu was incubated with both porcine pepsin and penicillopepsin in the presence of a lO-fold excess of C-leucine over Leu-Tyr-Leu. The products, leucine and leucylleucine, were separated by high voltage electrophoresis and analyzed for their specific radioactivity. At most, only traces of radio-... 

While the reaction with porcine pepsin (Figure 2) is qualitatively similar to that with penicillopepsin, there are obvious differences. First, hydrolysis predominated over transpeptidation and second, another trans-peptidation product, tri-leucine, was formed. The putative intermediate Leu-Leu-Tyr has, however, been detected. A detailed discussion of the significance of these experiments will be presented elsewhere. For the purpose of this review suffice it to say that they provide strong evidence for the involvement of an compulsory acyl intermediate in some pepsin and penicillopepsin catalyzed reactions. Hence, mechanistic proposals which do not involve acyl intermediates are insufficient for a complete description of pepsin catalyzed reactions. 

Our proposed mechanism is also compatible with the transpeptida-tion phenomenon which only occurs significantly for acid proteases at pH values greater than 4.0. Transfer of the proton from the carboxyl group of one hydrolysis product to the amino group of the other in Figure 8B could readily occur to produce R-COO" and H3 N-R. The transpeptidation products from the incubation of the peptide Leu-Try-Met with pepsin and penicillopepsin are the amino-trans-peptidation products Leu-Leu and Leu-Leu-Leu and the acyl-transpeptidation products Met-Met and Met-Met-Met (40). In order to produce the first products Leu-Leu and Leu-Leu-Leu, the tripeptide would bind initially such that the Leu-Trp bond is cleaved. Exchange of Trp-Met with a second Leu-Trp-Met is more facile than the exchange of the bound Leu product from the first hydrolysis. Transpep-... 

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