Effect of pH on pyrazole binding to liver alcohol dehydrogenase

1. Kinetic and equilibrium data have been determined at different pH between 4 and 10 for binding of the inhibitor pyrazole to liver alcohol dehydrogenase and to the binary complexes formed between enzyme and NADH or NAD+. 2. Pyrazole binding to free enzyme requires the protonated form of an ionizing group with a pKa of 9.2, agreeing with the pKa value reported for the water molecule bound at the catalytic zinc ion of the enzyme subunit. The rate of association of the inhibitor to the enzyme . NAD+ complex exhibits a similar pKa-7.6-dependence attributable to ionization of zinc-bound water in the latter binary complex. These observations lend support to the idea that pyrazole combines to the catalytic zinc ion on complex formation with the enzyme, zinc-bound water most likely being displaced by the inhibitor. 3. The rate of dissociation of the inhibitor from the ternary enzyme . NAD+ . pyrazole complex is proportional to the hydrogen ion concentration over the examined pH range (4-8). This effect of pH, which is proposed to reflect ionization of the enzyme-bound inhibitor with a pKa value below 4 (indirectly estimated to 2.4), accounts for the exceptional stability of the ternary complex at neutral and alkaline pH. It is concluded that pyrazole, by analogy to water and alcohol ligands, undergoes a drastic pKa perturbation on binding to the catalytic zinc ion in the enzyme . NAD+ complex.