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Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease.
Biochem J. 1999 May 15; 340 (Pt 1):113-7.BJ

Abstract

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 (kcat/Km=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, kinact/Ki=208200+/-36000 M-1. s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l- prolin e, kinact/Ki=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.

Authors+Show Affiliations

Department of Pathology, University of California, San Francisco, CA, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10229665

Citation

Chan, V J., et al. "Expression and Alteration of the S2 Subsite of the Leishmania Major Cathepsin B-like Cysteine Protease." The Biochemical Journal, vol. 340 (Pt 1), 1999, pp. 113-7.
Chan VJ, Selzer PM, McKerrow JH, et al. Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease. Biochem J. 1999;340 (Pt 1):113-7.
Chan, V. J., Selzer, P. M., McKerrow, J. H., & Sakanari, J. A. (1999). Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease. The Biochemical Journal, 340 (Pt 1), 113-7.
Chan VJ, et al. Expression and Alteration of the S2 Subsite of the Leishmania Major Cathepsin B-like Cysteine Protease. Biochem J. 1999 May 15;340 (Pt 1):113-7. PubMed PMID: 10229665.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease. AU - Chan,V J, AU - Selzer,P M, AU - McKerrow,J H, AU - Sakanari,J A, PY - 1999/5/7/pubmed PY - 1999/5/7/medline PY - 1999/5/7/entrez SP - 113 EP - 7 JF - The Biochemical journal JO - Biochem J VL - 340 (Pt 1) N2 - 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 (kcat/Km=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, kinact/Ki=208200+/-36000 M-1. s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l- prolin e, kinact/Ki=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. SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/10229665/Expression_and_alteration_of_the_S2_subsite_of_the_Leishmania_major_cathepsin_B_like_cysteine_protease_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/10229665/ DB - PRIME DP - Unbound Medicine ER -