Catalytic significance of binary enzyme-aldehyde complexes in the liver alcohol dehydrogenase reaction.
The interaction of liver alcohol dehydrogenase with NADH and aldehyde substrates has been characterized with respect to ternary-complex formation by the apparently non-preferred pathway which involves intermediate formation of binary enzyme X aldehyde complexes. Rate constant estimates are reported for dimethylaminocinnamaldehyde (DACA) binding to free enzyme and for NADH binding to the enzyme X DACA complex. The rate of NADH (or NAD+) association to liver alcohol dehydrogenase is not detectably affected by DACA binding to the enzyme, but the NADH dissociation rate decreases approximately by a factor of 6. The NADH-induced increase in affinity of the enzyme for DACA is similarly attributable to a decreased dissociation rate rather than an increased association rate of the aldehyde. DACA dissociates much more rapidly than coenzyme from the enzyme X NADH X aldehyde complex and shows a higher association rate constant than NADH in its interaction with free enzyme. It is concluded from these results that the enzymic reduction of typical aldehyde substrates will conform to a rate equation which is experimentally indistinguishable from that of a compulsory-order mechanism with coenzyme binding preceding substrate binding, and that this rate equation will obtain irrespective of which pathway for ternary-complex formation is actually preferred. Rate equations provide no reliable information about the order of ligand binding in ternary-complex systems. A flow analysis is presented which indicates that coenzyme and substrate are actually bound in random order to liver alcohol dehydrogenase during the enzymic reduction of aldehydes by NADH. The enzyme X aldehyde pathway for ternary-complex formation is fully kinetically competent, and reaction flow via this pathway may predominate when aldehyde concentrations exceed those required for half-saturation of free enzyme. Binary enzyme X aldehyde complexes are seemingly insignificant with respect to the rate behaviour of the enzyme, but may provide most significant and even predominant contributions to the catalytic reaction flow.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002934:Cinnamates / Q000378:metabolism / Q000494:pharmacology; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010783:Photometry; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D000447:Aldehydes; D002934:Cinnamates; D046911:Macromolecular Substances; D009243:NAD; C018523:4-dimethylaminocinnamaldehyde; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase