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Biochem. J. (1999) 340 (113–117) (Printed in Great Britain)

Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease

Victor J. CHAN*†, Paul M. SELZER*†1, James H. McKERROW*†‡ and Judy A. SAKANARI*†2

*Department of Pathology, University of California, San Francisco, CA, U.S.A., †Veterans Administration Medical Center, San Francisco, CA, U.S.A., and ‡Department of Pharmaceutical Chemistry, Univerity of California, San Francisco, CA, U.S.A.

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly²³⁴ at the S₂ subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly²³⁴ to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (k_cat/K_m = 3740±413 M^-1·s^-1 versus 472±72.4 M^-1·s^-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, k_inact/K_i = 208200±36000 M^-1·s^-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)--isoleucyl-L-proline, k_inact/K_i = 199200±32900 M^-1·s^-1], support the findings that this protozoan protease has the P₂ specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.

¹ Present Address: I. Frauenklinik, Ludwig-Maximilians Universitat, Munich, Germany, and Hoechst Roussel Vet GmbH Research Pharmaceuticals, Frankfurt, Germany.

² To whom correspondence should be addressed at Department of Biology, Sonoma State University, Rohnert Park, CA 94928-3609, U.S.A. (e-mail jsak@itsa.ucsf.edu).

Received 30 November 1998/13 January 1999; accepted 18 February 1999

The Biochemical Society, London © 1999

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