Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

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Abstract
The coordination environment of the catalytically active metal ion of horse liver alcohol dehydrogenase was investigated by EPR methods with use of the active-site-specific CO2+-reconstituted enzyme. The EPR absorption spectrum of the metal-substituted enzyme is characteristic of a rhombically distorted environment. The spectrum of the enzyme-NAD+ complex shows approximate axial symmetry of the metal ion site, indicating that binding of the coenzyme induces a structural alteration in the active-site region. This environment is not significantly altered further by binding of the competitive inhibitor pyrazole. To assign the coordination number of the active-site metal ion, the zero-field splitting was determined on the basis of the temperature dependence of the spin-lattice relaxation of the Co2+ ion. The zero-field splitting energies are .apprxeq. 9 cm-1 for the free Co2+-reconstituted enzyme and .apprxeq. 46 and .apprxeq. 47 cm-1 for the enzyme-NAD+ and enzyme-NAD+-pyrazole complexes, respecitvely. On the basis of studies of structurally defined small molecule complexes, these values are compatible with a tetracoordinate metal ion in the active site of the free enzyme but a pentacoordinate metal ion in the binary enzyme-NAD+ complex and in the ternary enzyme-NAD+-inhibitor complex and, therefore, presumably also in the catalytically active ternary enzyme-NAD+-alcohol complex formed in the course of alcohol oxidation.