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J. Biol. Chem., Vol. 279, Issue 20, 21500-21510, May 14, 2004

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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**, and Toru Nakayama**

From the Protein Structure Section, Macromolecular Crystallography Laboratory, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702, ^¶Basic Research Program, Science Applications International Corp.-Frederick, NCI-Frederick, National Institute of Health, Frederick, Maryland 21702, the ^||Department of Biochemistry, School of Medicine, Kanazawa Medical University, Uchinada 1-1, Ishikawa 920-0293, Japan, the ^**Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 07 Aoba-yama, Sendai 980-8579, Japan, Suntory Institute for Bioorganic Research, Mishima-gun, Osaka 618-8503, Japan, and the Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan

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-Glu⁷⁸-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 Asp⁸² do not interact with each other; thus, the canonical catalytic triad is disrupted.

Received for publication, February 2, 2004 , and in revised form, March 1, 2004.

The atomic coordinates and structure factors (codes 1sn7, 1siu, and 1sio) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Cancer Institute, National Institutes of Health, Contract NO1-CO-12400. This work was also supported in part by a Grant-in-aid for Scientific Research (B), 15380072 from the Japan Society for the Promotion of Science (to K. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 301-846-5036; Fax: 301-846-6322; E-mail: wlodawer{at}ncifcrf.gov.

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