Binding of competitive inhibitors to the different pH-dependent forms of trypsin

Abstract

Some of the rotatory, spectral, ionic and catalytic properties of trypsin (EC 3.4.4.4) have been studied, in the pH range from 1.5 to 12.5, in the presence and absence of synthetic competitive inhibitors. In the alkaline and acidic pH ranges where trypsin is reversibly denatured, the formation of the trypsin-inhibitor complex protects the enzyme against transconformation; at neutral pH, where trypsin is in the native form, the complex formation sometimes induces a change in the enzyme structure. In the alkaline pH range (a) the rotatory dispersion curve of the complex is pH independent; (b) the formation of the complex induces a decrease of the enzyme absorbance; (c) the formation of the complex produces a proton uptake by the enzyme; (d) the affinity of inhibitors decreases when the pH increases. All these results can be related to the ionization of an enzyme group having an apparent pK of about 10. In the acidic pH range (a) the rotatory power of the complex is pH independent, and (b) the complex formation produces a proton release by the enzyme. The changes in the ionic and rotatory properties of trypsin as a function of inhibitor concentration have allowed the evaluation of the dissociation constant of the benzamidine-trypsin complex between pH 4.5 and 1.5. A scheme for the interaction between the inhibitor and the different pH dependent forms of trypsin is proposed: it accounts for the pH dependence of the complex dissociation constant and also for the dependence of the number of protons released by the enzyme. The lowering of the inhibitor affinity in the acidic pH range is due to the protonation of two enzyme groups, one having an apparent p K of 3.7, the other a true pK of 4.5. At neutral pH, the enzyme-inhibitor complex formation may induce a change in both the enzyme and the inhibitor structure; formation of the trypsin-proflavin complex, for instance, makes the inhibitor optically active.