Quenching of protein fluorescence by transient intermediates in the liver alcohol dehydrogenase reaction.
The addition of saturating concentrations of NAD-+ and alcohol to liver alcohol dehydrogenase in a stopped flow fluorimeter results in a triphasic quenching of enzyme fluorescence. A rapid quenching occurs with a rate constant of 300 to 500 s-minus 1, followed by a slower reaction at 50 to 100 s-minus 1, and ultimately followed by a very slow reaction. The addition of NAD-+ to enzyme in the absence of substrate causes a rapid quenching of enzyme fluorescence at 300 to 500 s-minus 1, with the same amplitude as the rapid phase in the presence of substrate. These studies demonstrate that NAD-+ binding to liver alcohol dehydrogenase causes a conformational change at a rate compatible with the previously reported rate constant for proton release, indicating that proton release is probably coupled to the conformational change.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D001592:Benzyl Alcohols / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D001592:Benzyl Alcohols; D009243:NAD; D000431:Ethanol; D000429:Alcohol Oxidoreductases