title: Mutation of active site serine residue with cysteine displays change in acyl-acceptor preference of β-peptidyl aminopeptidase from Pseudomonas aeruginosa PAO1.
abstract: A β-peptidyl aminopeptidase, a peptidase belonging to the P1 family, catalyzes aminolysis in accordance with its hydrolytic activity. We specifically examined β-peptidyl aminopeptidase of Pseudomonas aeruginosa PAO1 (BapF) to assess the effects of mutation of catalytic Ser with Cys or Thr on its catalytic ability. Recombinant BapF and its S237C mutant exhibited p-nitroaniline release activity toward β-homo-Gly-p-nitroanilide (βhGly-pNA), but the products of the enzyme reaction differed completely from one another. Wild-type BapF showed βhGly-βhGly-pNA synthetic activity, but the product vanished in a few minutes and converted to free βhGly. In contrast, the product βhGly-βhGly-pNA was synthesized by S237C BapF efficiently without degradation, indicating that because of the mutation, the enzyme came to recognize only the amine group as an acyl acceptor instead of water. Furthermore, a difference in acyl acceptor preference between that of wild type and S237C BapF was observed. When using cysteamine as an acyl acceptor, βhGly-cysteamine was synthesized only in the reaction using S237C BapF. In contrast, S237C BapF was unable to synthesize βhGly-cystamine when using cystamine as an acyl acceptor, although it was synthesized by wild-type BapF. Such a dynamic change in the acyl acceptor by the mutation of catalytic Ser with Cys is regarded as a unique feature of family P1 peptidases.
authors.affiliation: Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University, Tottori, 680-8553, Japan, arima@muses.tottori-u.ac.jp.
authors.forename: Jiro
authors.initials: J
authors.lastname: Arima
authors.forename: Ayumi
authors.initials: A
authors.lastname: Tanaka
authors.forename: Masazumi
authors.initials: M
authors.lastname: Morimoto
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Mori
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry*; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D012694:Serine
chemical_list: D012694:Serine; D000626:Aminopeptidases; D003545:Cysteine