Location of the binding site for chloride ion activation of cathepsin C.
Cathepsin C, a tetrameric lysosomal dipeptidyl-peptide hydrolase, is activated by chloride ion. The activation is shown here to be specific and pH-dependent, dissociation constants for chloride being lower at low pH. Bound chloride decreases the Km for the hydrolysis of chromophore labelled substrates without any significant change in Vmax, confirming its involvement in substrate binding. Determination of the kinetic parameters of chloride activation, using unlabelled substrates, has enabled its site of action to be located. The lower Km for the hydrolysis of simple amide substrates in the presence of Cl- shows that the S sites are involved. Possible involvement of the S' sites is excluded by the finding that the Km for the nucleophile in the transferase reaction is unaffected by chloride. The rates of inhibition by E-64 and iodoacetate are both chloride-dependent and, from the structure of the papain-E-64 complex, it is concluded that chloride binds close to the S2 site. The binding of guanidinium ion, a positively charged inhibitor, to the S site is dependent on chloride. Based on these results, a model is proposed to explain the chloride activation of cathepsin C. The possible physiological role of chloride in the regulation of proteolysis in the lysosome is discussed.
Cigic
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B
Department of Biochemistry, Jozef Stefan Institute, Ljubljana, Slovenia. blaz.cigic@ijs.si
Pain
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RH
D001665:Binding Sites; D020789:Cathepsin C; D002712:Chlorides / Q000378:metabolism / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008954:Models, Biological; D013379:Substrate Specificity
D002712:Chlorides; D015853:Cysteine Proteinase Inhibitors; D007461:Iodoacetates; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C; D007930:Leucine; D019791:Guanidine; C024974:E 64