TEMPORARY INHIBITION OF PAPAIN BY HAIRPIN LOOP MUTANTS OF CHICKEN CYSTATIN

Temporary inhibition of the cysteine proteinases papain and cathepsin L was observed with several hairpin loop mutants of recombinant chicke n cystatin at enzyme concentrations above nanomolar. Kinetic modelling of inhibition data, gel electrophoresis and amino acid sequencing rev ealed that reappearance of papain activity is due to selective cleavag e of the Gly(9)-Ala(10) bond in the N-terminal binding area of the chi cken cystatin variants, resulting in truncated inhibitors of lower aff inity. Cleavage of the same bond by contaminating papaya proteinase TV was ruled out by previous purification of papain and suitable control experiments. According to the proposed kinetic model, cleavage occurs within the enzyme-inhibitor complex with first order rate constants k (temp) of 2.3 x 10(-3) up to 5 x 10(-1) s(-1). A similar k(temp)/K-m r atio was found for all mutants (0.7 x 10(6) - 2.1 x 10(6) s(-1). M(-1) ); it is almost identical with the k(cat)/K-m ratio of the peptide sub strate Z-Phe-Arg-NHMec. These results suggest that distorted contacts of one of the hairpin loops affect binding of the N-terminal contact a rea in a way that covalent interaction of the Gly(9)-Ala(10) bond with the active-site Cys residue of papain can occur and the bond is cleav ed in a substrate-like manner.