ROLE OF CIS-TRANS ISOMERISM OF THE PEPTIDE-BOND IN PROTEASE SPECIFICITY - KINETIC STUDIES ON SMALL PROLINE-CONTAINING PEPTIDES AND ON POLYPROLINE

Aminopeptidase P, a proline-specific exopeptidase, was isolated from Escherichia coli, and the cis-trans specificity of its activity was critically tested by using l-phenylalanyl-l-proline, polyproline, and glycyl-L-prolyl-l-alanine as substrates, under conditions where a high ratio of enzyme activity to substrate concentration existed. The results of the study strongly suggest that aminopeptidase P, like prolidase [Lin, L.-N., & Brandts, J. F. (1979) Biochemistry 18, 43], can only hydrolyze the trans form of the X-L-Pro peptide bond, while the cis form has to isomerize before it can be cleaved. This study also shows that the isomeric specificity of aminopeptidase P is valuable for studying the conformation of proline-containing peptides preequilibrated in aqueous solution as well as in the solid form. The kinetic data for polyproline hydrolysis how clearly that the cis-to-trans isomerization of polyproline I, when dissolved in water, begins step by step from the N-terminal end rather than the C-terminal end, as suggested by others from NMR data. It was also found that the rate of isomerization for glycyl-l-prolyl-L-alanine in aqueous solution is ~ 16 times faster than that of glycyl-l-proline, suggesting that the tripeptide could be a better model for proline isomerism in proteins. Finally, the experimental results indicate that the isomeric states of the C-terminal proline residue in L-leucyl-l-phenylalanyl-l-proline can be determined by using two aminopeptidases in tandem: one of which (leucine aminopeptidase) cleaves the Leu-Phe bond and the other of which (prolidase) cleaves the Phe-Pro bond. © 1979, American Chemical Society. All rights reserved.