Competitive parabolic inhibition of bovine trypsin by benzamidines

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

A statistical treatment for solution of a family of simultaneous equations derived from enzyme inhibition studies

1. A techinique of simultaneous regression of a family of lines with any common intercept is described for the treatment of enzyme inhibition data. The method is iterative and is baed on minimiztion of a weighted of squares of all residues. 2. The weighting procedure may be varied according to experimental design; the simultaneous regression treatment provides the crossing point of all linear and angular coefficients, as well as estimates of the precision of fitted parameters. 3. There are no restrictions as to location of crossing points. The method can be applied to all known transformed functions, properly weighted, used in enzyme studies that result in straight lines. It is rather simple to use by non-kineticists and would be useful in preliminary diagnosis of inhibition types, prior to the application of nonlinear techniques which require knowledge of the model