Substrate recognition mechanism of prolyl aminopeptidase from Serratia marcescens

Molecular cloning of the gene and the crystal structure of the prolyl amino peptidase [EC 3.4.11.5] from Serratia marcescens have been studied by us [J . Biochem. 122, 601-605 (1997); ibid. 126, 559-565 (1999)]. Through these s tudies, Phe139, Tyr149, Glu204, and Arg136 were estimated to be concerned w ith substrate recognition. To elucidate the details of the mechanism for th e substrate specificity, the site-directed mutagenesis method was applied. The F139A mutant showed an 80-fold decrease in catalytic efficiency (k(cat) /K-m) but the Y149A mutant did not show a significant change in catalytic e fficiency. The catalytic efficiency of the E204Q mutant was about 4% of tha t of the wild type. The peptidase activity of the mutant (R136A) was marked ly decreased, however, arylamidase activity with Pyr-PNA was retained as in the wild-enzyme. From these results, it was clarified that the pyrrolidine ring and the amino group of proline at the S1 site were recognized by Phe1 39 and Glu204, respectively. P1' of a substrate was recognized by Arg136. O n the other hand, the enzyme had two cysteine residues. Mutants C74A and C2 71A were inhibited by PCMB, but the double mutated enzyme (C74/271A) was re sistant to it.