Cleavage of the X-Pro peptide bond by pepsin is specific for the trans isomer.

Abstract

Fluorine nuclear magnetic resonance studies of the cleavage of peptides containing a 4-fluorophenylalanine (FPhe)-Pro bond have been performed in order to determine the conformational specificity of FPhe-Pro bond cleavage by pepsin. The peptides selected were substrates of HIV protease or of avian sarcoma virus protease, both of which have been reported to be cleaved specifically at X-Pro by pepsin as well as by the corresponding viral protease enzyme. By working at 0 degrees C, it was possible to separate kinetically cleavage and cis/trans isomerization. For the case of the protease substrate, Ser-Gln-Asn-FPhe-Pro-Ile-Val-Gln, cleavage was shown to be specific for the trans conformation. A value for the rate constant for hydrolysis of the trans peptide divided by the Michaelis constant, ktH/KMtrans = 0.3 min-1 mM-1 was obtained with this substrate, and the Michaelis constant appears to be considerably higher than the substrate concentration, 3.7 mM, used in the study. On a slower time scale, additional cleavages can readily be detected. For the avian leukemia virus protease substrate, Thr-Phe-Gln-Ala-FPhe-Pro-Leu-Arg-Glu-Ala, the cleavage was both slower and less specific. In addition to the primary cleavage at the FPhe-Pro site, cleavage also occurs at the Ala-FPhe bond on a somewhat slower time scale. In addition to the conformational specificity of the cleavage reaction, these results indicate that pepsin is a better model for HIV protease than for avian leukemia virus protease.