THE AFFINITY-LABELING OF CATHEPSIN-S WITH PEPTIDYL DIAZOMETHYL KETONES - COMPARISON WITH THE INHIBITION OF CATHEPSIN-L AND CALPAIN

Since peptidyl diazomethyl ketones are useful irreversible inhibitors for inactivating cysteinyl proteinases in vitro and in vivo and in ord er to reveal their role, we set out to obtain selective and effective reagents for cathepsin S. A number of such derivatives with hydrophobi c amino acid residues, such as valine, leucine and tryptophane in posi tions adjacent to the primary specificity site were synthesized and th ese provided inhibitors rapidly acting at high dilution. For example, 1 nM Z-Leu-Leu-Nle-CHN2 inactivates cathepsin S with k2nd = 4.6 x 10(6 ) M-1 . s-1 at pH 6.5, 25-degrees-C. Similarities to the specificities of cathepsin L and calpain were evident. However, Z-Val-Val-NleCHN2 i s over 300 times more effective in inactivating S than L. On the other hand, Z-Phe-Tyr(t-Bu)CHN2 is about 10(4) more effective against L tha n S. Reagents are thus now available for a clear discrimination betwee n these proteases.