Determination of the equilibrium distibution between alcohol and aldehyde substrates when bound to horse liver alcohol dehydrogenase using magnetic resonance.
The interactions of horse liver alcohol dehydrogenase (LADH) with the rho-trifluoromethyl derivatives of benzyl alcohol, benzaldehyde, and benzoic acid have been investigated by use of 19F nuclear magnetic resonance. The aldehyde and alcohol are good substrates for the enzyme and display kinetic characteristics similar to other alcohol/aldehyde pairs. In single-turnover experiments with NADH, rho-(trifluoromethyl)benzaldehyde shows biphasic kinetics similar to those shown by other aromatic aldehydes, which led Bernhard and co-workers [Bernard, S. A., Dunn, M. F., Luisi, P. L., & Shack, P. (1970) Biochemistry 9, 185] to propose half-of-the-sites reactivity for this enzyme. Fluorine magnetic resonance demonstrates that under equilibrium conditions at 4 degrees C and pH 8.75 rho-(trifluoromethyl)benzyl alcohol binds to LADH with a dissociation constant of 1 x 10(-3) M. Addition of 1 equiv of NAD to a stoichiometric complex of LADH and the alcohol generates a discrete resonance. This resonance corresponds to the ternary complex of LADH, NAD, and alcohol with little contribution from the NADH and aldehyde ternary complex. Quantitation shows that the bound NAD-alcohol pair is favored by at least a factor of 20 over the bound NADH-aldehyde pair at equililbrium. These results are consistent with explanations for the biphasic kinetics which involve half-of-the-sites reactivity of multiple steps in the reaction of each catalytic site. These results eliminate explanations for the biphasic kinetics of this system which require this equilibrium constant to have a value near unity or those which give greatest thermodynamic stability to the heterodimer LADH [(NAD-alcohol)(NADH-aldehyde)].
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001547:Benzaldehydes; D001565:Benzoates; D001592:Benzyl Alcohols; D006736:Horses; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D013379:Substrate Specificity; D014050:Toluene / Q000031:analogs & derivatives*
D001547:Benzaldehydes; D001565:Benzoates; D001592:Benzyl Alcohols; D008024:Ligands; C028385:4-(trifluoromethyl)benzyl alcohol; D014050:Toluene; C028384:4-(trifluoromethyl)benzaldehyde; C028383:4-(trifluoromethyl)benzoic acid; D000429:Alcohol Oxidoreductases