HAIRPIN LOOP MUTATIONS OF CHICKEN CYSTATIN HAVE DIFFERENT EFFECTS ON THE INHIBITION OF CATHEPSIN-B, CATHEPSIN-L AND PAPAIN

Five recombinant hairpin loop variants of chicken cystatin (Delta V55, Delta V55-S56, Delta P103-L105, Delta I102-Q107, loop2-KD2) were cons tructed by cassette mutagenesis, expressed in E. coli, purified to hom ogeneity, characterized by protein-chemical means and by their inhibit ory properties. The variant forms, modified in two of the three postul ated cysteine proteinase binding regions, were inhibitorily active. Ho wever, the equilibrium dissociation constants of the complexes between papain as well as human cathepsin B or L and the cystatin variants sh ow a weaker affinity for all three enzymes compared with recombinant c hicken cystatin. These results prove the contribution of both hairpin loops to complex formation with the three enzymes. Furthermore, the ki netic constants indicate discrete differences in the molecular mechani sm of interaction between chicken cystatin and papain, cathepsin B, an d cathepsin L. Inhibition of cathepsin L was much less affected than i nhibition of papain or cathepsin B by the modifications achieved in th e five variants. Remarkably, at high enzyme concentration (above 0.5 n M) inhibition of papain by these variants was 'temporary', that means, active papain was released from the enzyme-inhibitor complex within m inutes to hours (compare [I]).