Acyl and amino intermediates in penicillopepsin-catalysed reactions and activation by nonsubstrate peptides

Abstract

The action of penicillopepsin [Penicillium janthinellum] 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 1st 2 and Tyr-Tyr from the 3rd substrate was observed. These reactions presumably proceed via a covalent amino-intermediate in analogy to pig pepsin. With penicillopepsin the yields of transpeptidation are low. 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, 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 small effects on hydrolysis. The activators increase kcat [catalytic rate constant] but have no effect on Km. Studies of the cleavage of 6 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 .mu.M), is an activator for the cleavage of Leu-Tyr-NH2 (Ka = 500 .mu.M). Pepstatin, an inhibitor of the proteolytic activity of acid proteases, inhibits the action of penicillopepsin on Leu-Tyr-NH2. The action of penicillopepsin on small peptides is probably qualitatively similar to that of pig pepsin. Transpeptidation reactions of the amino acid and the acyl transfer type were observed. There are considerable differences in the effects of pH, and in relative specificity between the 2 enzymes.