The pH variation of steady-state kinetic parameters of site-specific Co(2+)-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 kcat and kcat/KM have been evaluated under initial velocity conditions for the native and the active site-specific Co(2+)-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 kcat (pK1 congruent to 6.7, pK2 congruent to 10.6) belong to the ternary enzyme-NAD(+)-alcohol complex, whereas the ionizations governing kcat/KM (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 pKa congruent to 1.0) in the Co(2+)-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.
Maret
W
W
Department of Biochemistry and Molecular Biology, University of Chicago, Cummings Life Science Center, Illinois 60637.
Makinen
M W
MW
D000433:1-Propanol / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D019905:Benzyl Alcohol; D001592:Benzyl Alcohols / Q000378:metabolism; D003035:Cobalt / Q000737:chemistry*; D003511:Cyclohexanols / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D013696:Temperature; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry
D001592:Benzyl Alcohols; D003511:Cyclohexanols; D007477:Ions; D003035:Cobalt; D000431:Ethanol; D000433:1-Propanol; D000426:Alcohol Dehydrogenase; D015032:Zinc; D019905:Benzyl Alcohol