title: Site-directed mutagenesis of conserved Trp39 in Rhizomucor pusillus pepsin: possible role of Trp39 in maintaining Tyr75 in the correct orientation for maximizing catalytic activity.
abstract: Replacement of Trp39 of Rhizomucor pusillus pepsin (RMPP) by Asn or Cys resulted in a marked decrease in the milk-clotting and proteolytic activities. Kinetic analysis with chromogenic synthetic oligopeptides as substrates revealed that the mutations caused marked changes in the kcat value, but only slight changes in the Km value. Similar enzymatic properties were observed in mutants of Tyr75, which was shown to have a role in enhancing the catalytic activity. Both Tyr75Asn and Trp39Asn mutants rapidly lost the activity at high temperatures due to autocatalytic digestion at two sites. The structures of several aspartic proteinases including RMPP, as revealed by X-ray crystallographic studies, showed that Trp39 occupies a position close to Tyr75 and the N delta atom of Trp39 within hydrogen-bonding distance of the hydroxyl side chain of Tyr75. These observations suggest that Trp39 plays a role in maintaining Tyr75 in the correct orientation in aspartic proteinases, including RMPP.
authors.affiliation: Department of Biotechnology, University of Tokyo.
authors.forename: Y N
authors.initials: YN
authors.lastname: Park
authors.forename: J
authors.initials: J
authors.lastname: Aikawa
authors.forename: M
authors.initials: M
authors.lastname: Nishiyama
authors.forename: S
authors.initials: S
authors.lastname: Horinouchi
authors.forename: T
authors.initials: T
authors.lastname: Beppu
mesh_terms: D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence; D004795:Enzyme Stability; D006358:Hot Temperature; D008958:Models, Molecular; D009090:Mucorales / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
chemical_list: D014364:Tryptophan; D016282:Aspartic Acid Endopeptidases