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 (Val10Arg cystatin C), the inhibition constant, K(i), increased 31-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 K(i) was less than 2-fold. In a similar study, using cathepsin B in which the substitution to Gln is instead at Glu-171, no such difference in how K(i) is affected was observed. Both Glu-245 and Glu-171 are located in the S2 subsite of cathepsin B. The observed effects on K(i) indicate that the additional positive charge introduced in Val10Arg 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 K(i) value of 0.13 nM was obtained for the interaction of Val10Arg cystatin C with papain.(ABSTRACT TRUNCATED AT 250 WORDS)
Lindahl
P
P
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
Ripoll
D
D
Abrahamson
M
M
Mort
J S
JS
Storer
A C
AC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000378:metabolism*; D003198:Computer Simulation; D055316:Cystatin C; D015891:Cystatins / Q000737:chemistry / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010206:Papain / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D014633:Valine / Q000378:metabolism*
C525030:CST3 protein, human; C525032:Cst3 protein, rat; D055316:Cystatin C; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D009838:Oligodeoxyribonucleotides; D002401:Cathepsin B; D010206:Papain; D014633:Valine