Spectral evidence for three metal-linked ionization equilibria in the interaction of cobalt(II) horse liver alcohol dehydrogenase with coenzyme and substrate.
The visible absorption bands in the region 525-575 nm of the catalytic cobalt ion in cobalt(II) horse liver alcohol dehydrogenase show characteristic pH-dependent changes both in the free enzyme and its complexes with nicotinamide adenine dinucleotide (NAD+) and NAD+ plus ethanol or 2,2,2-trifluoroethanol. In the free enzyme, the change of the coordination environment has an apparent pK of about 9.4. In the binary complex with NAD+ the spectral changes are complex, indicating changes in the coordination sphere in a lower pH range with an estimated pK value of about 7.9. The ternary complexes enzyme X NAD+ X ethanol and enzyme X NAD+ X 2,2,2-trifluoroethanol exhibit very similar, characteristic spectral features; their apparent pK values are 6.3 and less than 4, respectively. We ascribe these pK values to the ionization of the alcohol bound in the ternary complexes. The results demonstrate that the catalytic cobalt ion is sensing changes of the ionization state of the protein when going from low pH forms to high pH forms both in the absence and presence of coenzyme and substrate/inhibitor.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D003035:Cobalt / Q000378:metabolism*; D000431:Ethanol / Q000378:metabolism; D006736:Horses; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D013053:Spectrophotometry
D009243:NAD; D003035:Cobalt; D000431:Ethanol; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase