title: Substrate recognition mechanism of prolyl aminopeptidase from Serratia marcescens.
abstract: Molecular cloning of the gene and the crystal structure of the prolyl aminopeptidase [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 studies, Phe139, Tyr149, Glu204, and Arg136 were estimated to be concerned with substrate recognition. To elucidate the details of the mechanism for the 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 efficiency. The catalytic efficiency of the E204Q mutant was about 4% of that of the wild type. The peptidase activity of the mutant (R136A) was markedly decreased, however, arylamidase activity with Pyr-bNA 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 Phe139 and Glu204, respectively. P1' of a substrate was recognized by Arg136. On the other hand, the enzyme had two cysteine residues. Mutants C74A and C271A were inhibited by PCMB, but the double mutated enzyme (C74/271A) was resistant to it.
authors.affiliation: School of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi, Nagasaki 852-8521, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Ito
authors.forename: T
authors.initials: T
authors.lastname: Inoue
authors.forename: T
authors.initials: T
authors.lastname: Kabashima
authors.forename: N
authors.initials: N
authors.lastname: Kanada
authors.forename: H S
authors.initials: HS
authors.lastname: Huang
authors.forename: X
authors.initials: X
authors.lastname: Ma
authors.forename: N
authors.initials: N
authors.lastname: Azmi
authors.forename: E
authors.initials: E
authors.lastname: Azab
authors.forename: T
authors.initials: T
authors.lastname: Yoshimoto
mesh_terms: D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011392:Proline / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000187:drug effects; D012706:Serratia marcescens / Q000201:enzymology*; D013379:Substrate Specificity; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology
chemical_list: D010455:Peptides; D020245:p-Chloromercuribenzoic Acid; D011392:Proline; D000626:Aminopeptidases; C022450:prolyl aminopeptidase; D003545:Cysteine