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The inhibitory effects of proline-containing peptides and their derivatives on prolyl endopeptidases from Flavobacterium meningosepticum and bovine brain were compared. Replacement of the carboxyl terminal proline in N-blocked peptides with prolinal resulted in remarkable decreases in Ki values for both prolyl endopeptidases. Further reduction of the prolinal to prolinol led to a decrease in their inhibitory effects. Z-Pro-, Z-Val-, and Suc-Pro-prolinals were similarly inhibitory for both the enzymes with Ki values of nM order. However, the inhibitory effects of Z-Pyr-prolinal and Boc-Pro-prolinal on these enzymes were significantly distinguished: they strongly inhibited the mammalian prolyl endopeptidase with Ki values of nM order, while the Ki values of these compounds for the microbial enzyme were only of microM order. These results suggest that there are some structural differences in the S2 and S3 subsites between the two enzymes, though their substrate specificities are apparently indistinguishable.

Microorganisms capable of producing L-pyrrolidonecarboxylate peptidase [L-pyrrolidonyl peptidase, EC 3.4.11.8] were screened and a strain of Bacillus amyloliquefaciens was chosen as one of the most potent producers of the enzyme. The enzyme was purified from lysozyme-lysate of the bacterial cells by salting out with ammonium sulfate, adsorption on DEAE-cellulose, covalent chromatography on PCMB-Sepharose and by gel filtration on Sephadex G-150. By these procedures, the enzyme was purified about 800-fold with an activity recovery of 9%, and the preparation was electrophoretically homogenous. The enzyme was most active and stable at pH 7-8. The presence of 2-mercaptoethanol and EDTA was effective for stabilizing the enzyme. The molecular weight was estimated to be 72,000 by the gel filtration method and to be 24,000 by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a subunit oligomer, presumably trimer. The enzyme was inactivated by the addition of PCMB, sodium tetrathionate, Hg2+ and Cu2+, but the activity lost was restored by the addition of 2-mercaptoethanol and EDTA. The purified enzyme split amide and ester linkages in L-pyroglutamyl derivatives of L-alanine, beta-naphthylamine, alpha-naphthol, and 4-methylumbelliferone, but was completely inert towards various peptides and esters used as substrates for usual amino- and carboxy-peptidases, and for endopeptidases such as trypsin, subtilisin and alpha-chymotrypsin.

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