The nature of binding of competitive inhibitors to alcohol dehydrogenases.

Two classes of metal ion binding sites in horse liver alcohol dehydrogenase are distinguished by the observed rates of replacement of zinc by cobalt in acetate and in Z-(N-morpholine)-ethane sulfonic acid buffers. Hybrid enzymes containing both zinc and cobalt metal ions have been prepared and exhibit visible absorption maxima at 655 nm and 740 nm. Binding of azide, a substrate-competitive inhibitor, to yeast alcohol dehydrogenase and to native and metallo hybrid horse liver alcohol dehydrogenases has been investigated. The infrared absorption maximum for azide bound to yeast alcohol dehydrogenase in the presence of NAD+ is 2070 cm-l, and that for azide bound to native horse liver alcohol dehydrogenase is 2065 cm-l. The frequency of azide in a solution of a hybrid liver enzyme is within experimental error equal to that observed upon binding to the zinc enzyme. Kinetic analysis also indicates that the binding of azide to zinc and hybrid liver enzymes is similar. No change is observed in the visible spectrum of the hybrid enzyme upon binding of azide. No change in the visible spectrum is observed upon binding of pyrazole, a neutral substrate-competitive inhibitor, to hybrid horse liver alcohol dehydrogenase. Stopped flow measurements show that the rate of binding of pyrazole to the hybrid enzyme is at least as great as, and perhaps greater by a factor of two than, binding to the native enzyme. Similarly, 1, IO-phenanthroline binds twice as fast to the hybrid enzyme as to the zinc enzyme, and no change in the visible spectrum is observed upon binding of this coenzyme-competitive inhibitor. These results may be interpreted to indicate that none of these inhibitors binds to the “easily exchangeable” metal ions in horse liver alcohol dehydrogenase.