The consequences of nucleotide binding to liver alcohol dehydrogenase.
Extensive and informative steady-state kinetic investigations of the mechanisms of horse liver alcohol dehydrogenase have recently been complemented by observations of the fluorescence and spectroscopic characteristics of transient intermediates by rapid-reaction techniques. In this way it was possible to study separately steps involved during enzyme-substrate complex formation and during the catalytic process. It can be shown that a proton is liberated during complex formation before the transfer of a hydride ion from ethanol to form NADH. This must be due to a change in pK of a group on the enzyme protein and is linked to a change in tryptophan fluorescence. Pressure relaxation techniques have enabled us to study the rate constants of the change in tryptophan fluorescence linked to NAD+ binding and proton dissociation. We have shown that NAD+ binding occurs in two steps: a rapid secondorder association, followed by the substrate-induced isomerization to form the reactive enzyme-substrate intermediate. The isomerization rate constants were determined in both directions and their role in the overall reaction mechanism could be identified.
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009243:NAD; D010084:Oxidation-Reduction; D011485:Protein Binding
D009243:NAD; D000429:Alcohol Oxidoreductases