Expression and alteration of the S-2 subsite of the Leishmania major cathepsin B-like cysteine protease

The mature form of the cathepsin B-like protease of Leishmanin major (Lmajc atB) 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 hig h identity and structural similarity with cathepsin B, LmajcatB does not re adily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg- Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrol yse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzym es. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly(234) at the S-2 subsite of LmajcatB, which forms a la rger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastor is yeast expression system. The quality of the recombinant enzyme was confirm ed by kinetic characterization, N-terminal sequencing, and Western blot ana lysis. Alteration of Gly(234) to Glu, which is found at the corresponding s ite in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) ac tivity 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)-L-isoleucyl-L-proline, k(inact )/K-i = 199200+/-32900 M(-1.)s(-1)], support the findings that this protozo an protease has the P-2 specificity of cathepsin L-like enzymes while retai ning structural homology to mammalian cathepsin B,