Evidence for the interaction of valine-10 in cystatin C with the S2 subsite of cathepsin B

• The interactions between wild-type or mutant recombinant forms of human cystatin C and rat cathepsin B were characterized by measuring progress curves for substrate hydrolysis in the presence of inhibitor. The investigation was guided by the use of computer modeling and explores the possibility that amino acid residues in the N-terminal region of cystatin C interact with substrate-binding regions in the target enzyme. With cystatin C that has Val-10 replaced by an Arg residue (VallOArg cystatin C), the inhibition constant, Ki, increased 3 1-fold if the isosteric substitution Glu-245 to Gln was made in cathepsin B. When the wild-type form of the inhibitor was used, the corresponding effect on Ki was less than 2-fold. In a similar study, using cathepsin B in which the substitution to Gln is instead at Glu-17 1, no such difference in how Ki is affected was observed. Both Glu-245 and Glu-171 are located in the S2 subsite of cathepsin B. The observed effects on Ki indicate that the additional positive charge introduced in VallOArg cystatin C is interacting with the negative charge on Glu-245 in cathepsin B when these two proteins form a complex; the cystatin variant is thus binding in a substratelike manner with this region of the enzyme. Indirectly, these results suggest that when native cystatin C and cathepsin B form a complex, Val-10 in the inhibitor interacts with the S2 subsite of the enzyme. A Ki value of 0.13 nM was obtained for the interaction of VallOArg cystatin C with papain. Compared to the dissociation equilibrium constant for wild-type cystatin C and papain, this value represents a 12 000-fold decrease in affinity. The corresponding effect with cathepsin B was only 15-fold, which presumably reflects the importance of the P2 position in binding of the N-terminal region of cystatin C to different cysteine proteinases.

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NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

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