Crystallographic and biochemical investigations of kumamolisin-As, a serine-carboxyl peptidase with collagenase activity

Alexander Wlodawer, Mi Li, Alla Gustchina, Naoki Tsuruoka, Masako Ashida, Hiroyuki Minakata, Hiroshi Oyama, Kohei Oda, Tokuzo Nishino, Toru Nakayama

研究成果: Article査読

37 被引用数 (Scopus)

抄録

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 (Ser278-Glu 78-Asp82) 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 Ser278, His78, and Asp 82 do not interact with each other; thus, the canonical catalytic triad is disrupted.

本文言語English
ページ(範囲)21500-21510
ページ数11
ジャーナルJournal of Biological Chemistry
279
20
DOI
出版ステータスPublished - 2004 5 14

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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