BINDING TO THERMOLYSIN OF PHENOLATE-CONTAINING INHIBITORS NECESSITATES A REVISED MECHANISM OF CATALYSIS

Competitive inhibition as a function of pH for the metalloendoprotease thermolysin by derivatives of L-alpha(-(2-hydroxyphenyl)benzenepropanoyl-L-tryptophanlylglycine exhibits a diagnostic bell shape. Binding is maximal between two pK(a) values: on the acidic limb the apparent K-1 value is regulated by an unchanging enzymic ionization (pK(a) 5.3) which is also seen in the substrate-hydrolysis kinetics (k(cat.)/K-m) whereas the alkaline limb for inhibition varies and depends specifically on the pK(a) of the phenolic group in the inhibitor. Although it should be the phenolate form of the inhibitor that co-ordinates more efficiently to the active-site Zn2+, the apparent K-1 shifts from pH-independent at pH values immediately below the inhibitor's pk(a), to progressively weaker binding at higher pH. This is explained by an anomalous acidity for the exchangeable solvent molecule that is attached to enzymic Zn2+ in the absence of substrate or inhibitor. Since OH- cannot be displaced from the enzyme as readily as H2O, a compensating pK(a) of 5.3 possessed by Zn2+-bound water rationalizes the binding characteristics, yielding the level pH profile exhibited at intermediate pH values. Recognition of the implicit heightened Lewis acidity of the metal ion in thermolysin leads to a revision of the mechanism of catalysis. The substrate amide bond becomes-activated for hydrolysis by carbonyl-group co-ordination to the especially acidic Zn2+ ion (completely displacing the H2O/OH- species otherwise bound). The imidazole group of enzymic residue His-231, also discerned in the pH profile for k(cat.)/K-m from its pK(a) of 8, provides general-base assistance for hydration of the activated scissile linkage in the first committed step of catalysis. Additional evidence from inhibition patterns shows how substrate-binding energy may be employed in this scheme to promote hydrolysis of peptides by thermolysin.