THE SYNTHESIS OF INHIBITORS FOR PROCESSING PROTEINASES AND THEIR ACTION ON THE KEX2 PROTEINASE OF YEAST

Peptidyl chloromethane and sulphonium salts containing multiple Arg an d Lys residues were synthesized as potential inhibitors of prohormone and pro-protein processing proteinases. The potencies of these compoun ds were assayed by measuring the kinetics of inactivation of the yeast Kex2 proteinase, the prototype of a growing family of eukaroytic prec ursor processing proteinases. The most potent inhibitor, Pro-Nvl-Tyr-L ys-Arg-chloromethane, was based on cleavage sites in the natural Kex2 substrate pro-alpha-factor. This inhibitor exhibited a K(i) of 3.7 nM and a second-order inactivation rate constant (k2/K(i)) of 1.3 x 10(7) M-1 . s-1 comparable with the value of k(cat)/K(m) obtained with Kex2 for the corresponding peptidyl methylcoumarinylamide substrate. The e nzyme exhibited sensitivity to the other peptidyl chloromethanes over a range of concentrations, depending on peptide sequence and alpha-ami no decanoylation, but was completely resistant to peptidyl sulphonium salts. Kinetics of inactivation by these new inhibitors of a set of 'c ontrol' proteinases, including members of both the trypsin and subtili sin families, underscored the apparent specificity of the compounds mo st active against Kex2 proteinase.