Two inhibitor molecules bound in the active site of Pseudomonas sedolisin: a model for the bi-product complex following cleavage of a peptide substrate Alexander Wlodawer, a, * Mi Li, a,b Alla Gustchina, a Hiroshi Oyama, c Kohei Oda, c Bret B. Beyer, d Jose Clemente, d and Ben M. Dunn d a Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA b Intramural Research Support Program, SAIC Frederick, National Cancer Institute at Frederick, Frederick, MD 21702, USA c Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan d Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA Received 6 December 2003 This paper is dedicated to Professor David Eisenberg on the occasion of his 65th birthday. Abstract High-resolution crystallographic analysis of a complex of the serine-carboxyl proteinase sedolisin with pseudo-iodotyrostatin revealed two molecules of this inhibitor bound in the active site of the enzyme, marking subsites from S3 to S3 0 . The mode of binding represents two products of the proteolytic reaction. Substrate specificity of sedolisin was investigated using peptide libraries and a new peptide substrate for sedolisin, MCA–Lys–Pro–Pro–Leu–Glu#Tyr–Arg–Leu–Gly–Lys(DNP)–Gly, was synthesized based on the results of the enzymatic and crystallographic studies and was shown to be efficiently cleaved by the enzyme. The kinetic pa- rameters for the substrate, measured by the increase in fluorescence upon relief of quenching, were: k cat ¼ 73  5s 1 , K m ¼ 0:12  0:011 lM, and k cat =K m ¼ 608  85 s 1 lM 1 . Published by Elsevier Inc. Keywords: Serine-carboxyl proteinases; Substrate specificity; Inhibitor binding; Reaction products Sedolisins (serine-carboxyl proteinases) form the family S53 of clan SB of serine proteinases, as annotated in the MEROPS database (http://merops.sanger.ac.uk). These enzymes have been known for a number of years as pepstatin-insensitive carboxyl proteinases [1]. Their placement in the same clan as subtilisin followed the solution of crystal structures of two representative members of this family, sedolisin (also known as Pseu- domonas serine-carboxyl proteinase or PSCP) [2,3] and kumamolisin (kumamolysin, KSCP) [4]. The nomen- clature describing these enzymes was changed several times in the last several years; the recently introduced terms “sedolisin” and “kumamolisin” [5] will be used throughout this paper. Sedolisins share a number of common properties such as maximum activity at comparatively low pH, the presence of conserved acidic residues (aspartate and glutamate) required for the activity, stabilization by a Ca 2þ cation, and the lack of inhibition by pepstatin. Pseudomonas sp. 101 sedolisin was the first member of the family to be isolated and described, and its enzymatic properties have been studied by biochemical and muta- genesis approaches [1,6–8]. Biochemical data are also available for several other related bacterial enzymes, such as sedolisin-B from Xanthomonas sp. T-22 (XSCP) [1]; kumamolisin, an enzyme isolated from a thermo- philic bacterium Bacillus novo sp. MN-32 [9]; kuma- molisin-B, an alcohol-resistant proteinase J-4 isolated from bacterium Bacillus coagulans [10]; and kumamoli- sin-As (ScpA), a peptidase with collagenolytic activity isolated from Alicyclobacillus sendaiensis [11]. Although neither the biological role of these enzymes in bacteria * Corresponding author. Fax: 1-301-846-6128. E-mail address: wlodawer@ncifcrf.gov (A. Wlodawer). 0006-291X/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.bbrc.2003.12.130 Biochemical and Biophysical Research Communications 314 (2004) 638–645 BBRC www.elsevier.com/locate/ybbrc