The action of penicillopepsin on a variety of small peptides was studied qualitatively and kinetically. With the substrate benzyloxycarbonyl-Phe-Leu, benzyloxycarbonyl-Tyr-Leu and acetyl-Phe-Tyr the formation of Leu-Leu from the first two and Tyr-Tyr from the third substrate was observed. These reactions presumably proceed via a covalent amino-intermediate in analogy to pig pepsin. However, with penicillopepsin the yields of transpeptidation are low. In contrast, transpeptidations via acyl transfer proceed in high yield with a variety of substrates. With Leu-Trp-Met both acyl and amino transfer occurs as shown by the formation of Leu-Leu and Met-Met; unlike with pig pepsin, however, no Leu-Leu-Leu or Met-Met-Met was observed. Nonsubstrate peptides activate the cleavage of small substrates. (The term 'cleavage' is used here to imply that the reaction is either a hydrolysis or a transpeptidation, or both. The term 'hydrolysis' will only be used for strictly hydrolytic reactions.) As with pig pepsin the activators increase predominantly the transpeptidation reaction and have only small effects on hydrolysis. The activators increase kcat but have no effect on Km. Studies of the cleavage of six different peptides show that at pH 4.7 Km is lower than at pH 3.4 while kcat is unaffected. As with pig pepsin activation by nonsubstrate peptides is greater at pH 4.7 than at pH 3.4. Benzyloxycarbonyl-Glu-Tyr which is an inhibitor of trypsinogen activation (Ki = 50 μM) is an activator for the cleavage of Leu-Tyr-NH2 (Ka = 500 μM). Pepstatin, an inhibitor of the proteolytic activity of acid proteases, also inhibits the action of penicillopepsin on Leu-Tyr-NH2.The major conclusion from these studies is that the action of penicillopepsin on small peptides is qualitatively similar to that of pig pepsin. Transpeptidation reactions of both the amino acid and the acyl transfer type have been observed. However, there are considerable differences in the effects of pH, and in relative specificity between the two enzymes.
The specificity of clostripain from Clostridium histolyticum. Mapping the S' subsites via acyl transfer to amino acid amines and peptides
