ABSTRACT
Competitive parabolic inhibition, a rare type of inhibition of one-substrate enzymes, is described for alpha-and beta-trypsin. The enzymes were so inhibited by two bis-benzamidines 4-4' diazoamino-bis-benzamidine, Berenil (DABB) and its platinum complex, DABB-PtCl2, acting on acyl-amino acid-peptidyl nitroanilides (Nan) substrates, when inhibitor concentrations exceed 10 mM and approch the millimolar range. The type of nonlinear inhibition observed require4s ternary complex formation between one enzyme molecule and two inhibitor molecules (M. E. M), and also permits the formation of the mixed ternary complex (M. E. S.). Binding of the first DABB molecule to the active center of trypsin takes place with K1 values of ca. 1.50 uM for both alpha-and- beta-trypsin. The secondary binding site binds the inhibitor with dissociation constants K12 close to 0.25 mM for both forms of the enzyme, as determined with different substrates. The dissociation constants of the ternary mixed complexes (Ksi and Kis), however, depend on the structural features of the substrates, which are of negligible importance for Bz-Arg-Nan, but significant for Ac-Phe-Arg-Nan and D-Val-Leu-Arg-Nan, reflecting subsite interactions between S1-S3 and S'2. Pentamidine, a diamidino-4,4'-diphenoxy-alkane with a flexible chain, behaved as a strict competitive inhibitor. This implies that the triazene moiety of DABB is involved in the interaction between the inhibitor and the secondary binding site of the enzyme