title: Crystallographic and biochemical investigations of kumamolisin-As, a serine-carboxyl peptidase with collagenase activity.
abstract: Kumamolisin-As (previously called ScpA) is the first known example of a collagenase from the sedolisin family (MEROPS S53). This enzyme is active at low pH and in elevated temperatures. In this study that used x-ray crystallographic and biochemical methods, we investigated the structural basis of the preference of this enzyme for collagen and the importance of a glutamate residue in the unique catalytic triad (Ser(278)-Glu(78)-Asp(82)) for enzymatic activity. Crystal structures of the uninhibited enzyme and its complex with a covalently bound inhibitor, N-acetyl-isoleucyl-prolyl-phenylalaninal, showed the occurrence of a narrow S2 pocket and a groove that encompasses the active site and is rich in negative charges. Limited endoproteolysis studies of bovine type-I collagen as well as kinetic studies using peptide libraries randomized at P1 and P1', showed very strong preference for arginine at the P1 position, which correlated very well with the presence of a negatively charged residue in the S1 pocket of the enzyme. All of these features, together with those predicted through comparisons with fiddler crab collagenase, a serine peptidase, rationalize the enzyme's preference for collagen. A comparison of the Arrhenius plots of the activities of kumamolisin-As with either collagen or peptides as substrates suggests that collagen should be relaxed before proteolysis can occur. The E78H mutant, in which the catalytic triad was engineered to resemble that of subtilisin, showed only 0.01% activity of the wild-type enzyme, and its structure revealed that Ser(278), His(78), and Asp(82) do not interact with each other; thus, the canonical catalytic triad is disrupted.
authors.affiliation: Protein Structure Section, Macromolecular Crystallography Laboratory, NCI-Frederick, National Institutes of Health, Frederick, MD 21702, USA. wlodawer@ncifcrf.gov
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
authors.forename: Mi
authors.initials: M
authors.lastname: Li
authors.forename: Alla
authors.initials: A
authors.lastname: Gustchina
authors.forename: Naoki
authors.initials: N
authors.lastname: Tsuruoka
authors.forename: Masako
authors.initials: M
authors.lastname: Ashida
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Minakata
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Oyama
authors.forename: Kohei
authors.initials: K
authors.lastname: Oda
authors.forename: Tokuzo
authors.initials: T
authors.lastname: Nishino
authors.forename: Toru
authors.initials: T
authors.lastname: Nakayama
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D017364:Collagenases / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D002268:Carboxypeptidases; D016282:Aspartic Acid Endopeptidases; C078894:kumamolysin; D017364:Collagenases