The inactivation of the cysteinyl exopeptidases cathepsin H and C by affinity-labelling reagents

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Abstract
An attempt has been made to extend to the cysteinyl exopeptidases cathepsin H and C affinity-labelling approaches shown to be effective with cysteinyl endopeptidases such as cathepsin B and L and the calcium-activated proteinase. This involved the preparation of amino acid and dipeptide derivatives with unblocked N-termini to satisfy the aminopeptidase and dipeptidylaminopeptidase characteristics of cathepsins H and C respectively. For covalent reactivity, the possibilities examined included diazomethanes (-CHN2), fluoromethanes (-CH2F) and dimethylsulphonium salt [-CH2S+(CH3)2]. A dipeptidylfluoromethane with a free amino group could not be prepared, perhaps due to inherent instability. Cathepsin H was inactivated by 1 .mu.M-H2N-Phe-CH2F (the ''H2N'' indicates a free unblocked amino group) (k2 = 1878 M-1 .cntdot. s-1); this reagent was without effect on cathepsins C and B, even at 100-fold this concentration. Analogous selectivity was shown by H2N-Ser(OBzl)-CHN2 and H2N-Phe-CH2S+(CH3)2, members of other classes of covalently binding reagents. For cathepsin C the dipeptide derivates H2N-Gly-Phe-CHN2 and H2N-Phe-Ala-CH2S+(CH3)2 caused rapid inactivation near 10-7 M. Hgiher concentrations inactivated cathepsin H and B, but the rates were slower by two to three orders of magnitude than for cathepsin C.