Evidence showing that a proline-specific endopeptidase has an absolute requirement for a trans peptide bond immediately preceding the active bond.

The proline-specific endopeptidase (EC 3.4.21.26) from Flavobacterium meningosepticum is specific for the cleavage of peptide bonds on the C-terminal side of prolyl residues. Such bonds will normally exist in the all-trans configuration. However, the preceding peptide bond in the sequence (i.e., on the N-terminal side of the prolyl residue) will exist as a mixture of cis and trans forms in solution. In this study, the activity of the proline-specific endopeptidase toward the substrates N-Cbz-Gly-Pro-MCA (where MCA = 4-methylcoumarinyl-7-amine) and N-Cbz-Gly-Pro-Leu-Gly has been examined. At a high ratio of enzyme activity/substrate concentration, the hydrolysis pattern for each substrate shows two well-separated kinetic phases. It is concluded that the fast kinetic phase, whose velocity depends on enzyme concentration, results from the direct hydrolysis of the active substrate bond (i.e., either the Pro-MCA or Pro-Leu bond, respectively) in molecules where the preceding Gly-Pro bond is trans. The slow phase, whose velocity is independent of enzyme concentration, is rate-limited by the cis-to-trans isomerization of those substrate molecules which initially have the preceding Gly-Pro bond in the cis configuration. That is, substrate molecules having the cis form of the Gly-Pro bond which precedes the active bond cannot be hydrolyzed directly but must first isomerize to the trans form before cleavage can occur. The amplitude, relaxation time, and activation energy for the slow phase are consistent with this interpretation. Thus, the proline-specific endopeptidase from Flavobacterium has an absolute requirement for a trans peptide bond at the position immediately preceding the active bond.