Catalytic significance of binary enzyme‐aldehyde complexes in the liver alcohol dehydrogenase reaction

Publisher Website
Google Scholar
Abstract
The interaction of [horse] liver alcohol dehydrogenase with NADH and aldehyde substrates was characterized with respect to ternary-complex formation by the apparently non-preferred pathway which involves intermediate formation of binary enzyme.cntdot.aldehyde complexes. Rate constant estimates are reported for dimethylaminocinnamaldehyde (DACA) binding to free enzyme and for NADH binding to the enzyme.cntdot.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 by a factor of .apprx. 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.cntdot.NADH.cntdot.aldehyde complex and shows a higher association rate constant than NADH in its interaction with free enzyme. The enzymic reduction of typical aldehyde substrates will probably conform to a rate equation which is experimentally indistinguishable from that of a compulsory-order mechanism with coenzyme binding preceding substrate binding, and 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 enzyme reduction of aldehydes by NADH. The enzyme.cntdot.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.cntdot.aldehyde complexes are seemingly insignificant with respect to the rate behavior of the enzyme, but may provide most significant and even predominant contributions to the catalytic reaction flow.