THE PH VARIATION OF STEADY-STATE KINETIC-PARAMETERS OF SITE-SPECIFIC CO2+-RECONSTITUTED LIVER ALCOHOL-DEHYDROGENASE - A MECHANISTIC PROBE FOR THE ASSIGNMENT OF METAL-LINKED IONIZATIONS

To identify ionizations of the active site metal-bound water in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1), the pH, solvent isotope, temperature, and anion dependences of the steady-state kinetic parameters k(cat) and k(cat)/K(M) have been evaluated under initial velocity conditions for the native and the active site-specific Co2+-reconstituted enzyme. In the oxidation of benzyl alcohol, a bell-shaped pattern of four prototropic equilibria was observed under conditions of saturating concentrations of NAD+. It is shown that the ionizations governing k(cat) (pK1 congruent-to 6.7, pK2 congruent-to 10.6) belong to the ternary enzyme-NAD+-alcohol complex, whereas the ionizations governing k(cat)/K(M) (pK1' congruent-to 7.5, pK2' congruent-to 8.9) belong to the binary enzyme-NAD+ complex. The ionizations pK1 and pK1' are not influenced by metal substitution and are ascribed to His-51 on the basis of experimental estimates of their associated enthalpies of ionization. On the other hand, pK2 and pK2' are significantly decreased (DELTA-pK(a) congruent-to 1.0) in the Co2+-enzyme and are attributed to the active site metal-bound water molecule. The shape of the pH profiles requires that the metal ion coordinates a neutral water molecule in the ternary enzyme-NAD+-alcohol complex under physiological conditions. The possible catalytic role of the water molecule within a pentacoordinate metal ion complex in the active site is discussed.