Influence of anions and pH on the conformational change of horse liver alcohol dehydrogenase induced by binding of oxidized nicotinamide adenine dinucleotide: binding of chloride to the catalytic metal ion.
The conformational change of horse liver alcohol dehydrogenase induced by binding of NAD+ was studied by electronic absorption spectroscopy using cobalt as a spectroscopic probe in the active site. The complex of the enzyme with NAD+ exists in an acidic and an alkaline form. The transition between the two forms proceeds through several intermediates and is controlled by an apparent pKa of 6.9. Only at pH values below this pKa can a complex between enzyme, NAD+, and Cl- be formed. The spectral changes indicate that chloride displaces the cobalt-bound water molecule in a tetracoordinate structure. We conclude that a negative charge at the active site is necessary to stabilize the closed conformation of the enzyme in the presence of NAD+. Spectral correlations are given which strongly support the postulation of a metal-bound alkoxide in the closed structure of the enzyme as an essential feature of the catalytic mechanism of horse liver alcohol dehydrogenase.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D000838:Anions; D004578:Electron Spin Resonance Spectroscopy; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry
D000838:Anions; D009243:NAD; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase