Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.
The coordination environment of the catalytically active metal ion of horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) has been investigated by electron paramagnetic resonance (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 approximately 9 cm-1 for the free Co2+-reconstituted enzyme and approximately 46 and approximately 47 cm-1 for the enzyme--NAD+ and enzyme--NAD+--pyrazole complex, respectively. 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.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003035:Cobalt / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D006736:Horses; D008099:Liver / Q000201:enzymology*; D009243:NAD; D011485:Protein Binding; D013696:Temperature
D009243:NAD; D003035:Cobalt; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase