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

Abstract

1. 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 . aldehyde complexes. Rate constant estimates are reported for dimethylaminocinnamaldehyde (DACA) binding to free enzyme and for NADH binding to the enzyme . DACA complex.2. The rate ofNADH (or NAD') association to liver alcohol dehydrogenaseis not detectably affected by DACA binding to the enzyme, but the NADH dissociation rate decreases approximately by a factor of 6. The NADHinduced 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. NADH . aldehyde complex and shows a higher association rate constant than NADH in its interaction with free enzyme.3. 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.4. 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. 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 . 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.Liver alcohol dehydrogenase [I ] catalyzes alcohol/aldehyde interconversion by a ternary-complex mechanism with NAD+/NADH as coenzymes. The enzyme shows a significant affinity for aldehyde substrates whether or not NADH is present (and vice versa) [1,2], which indicates that the productive enzyme. NADH . aldehyde complexes can be assumed to be formed by a basically random-order binding mechanism (Scheme 1). The enzyme. aldehyde pathway for ternary-complex formation in Scheme 1 does not appear to contribute appreciably to the catalytic reaction flow, however. The enzymic reduction of aldehydes by NADH invariably has been found to conform to the rate equation of a compulsoryorder mechanism with coenzyme binding preceding binding of the aldehyde substrate [3 -61.The reason why this binding order appears to be preferred remains obscure. Essentially no information is available about the effect of coenzyme on the kinetics of substrate binding, or about th...

Analysis of the mechanism of chloramphenicol acetyltransferase by steady-state kinetics. Evidence for a ternary-complex mechanism

“…However, the use of different acylacceptors, in the forward reaction, has led to the suggestion that acetyl-CoA binds before chloramphenicol. Andersson et al (1984) have reconciled similarly conflicting observations for liver alcohol dehydrogenase. Both substrates bind independently of one another, but, under the conditions normally chosen for kinetic analysis, the E-NADH pathway to the ternary complex is preferred.…”

Mechanistic origin of the sigmoidal rate behaviour of glucokinase

“…There is strong reason to question the validity of the latter argument, because theoretical analyses have failed to reveal any intuitively obvious relationship between the rate behaviour of and partitioning of reaction flow in random ternary-complex systems (Pettersson, 1969;Andersson et al, 1984). The present paper draws attention to this interpretational complication and shows that the basic kinetic properties of glucokinase can be accounted for without the assumption that glucose interacts with distinct conformational states of free enzyme.…”

Alternative pathways and reactions of benzyl alcohol and benzaldehyde with horse liver alcohol dehydrogenase

“…Random Pathways. Although the kinetic mechanism of alcohol dehydrogenase is described as compulsory or preferred ordered, it has been suggested that steady-state kinetics is inadequate to establish the extent of reaction by alternative pathways (Andersson et al, 1984a). A random pathway has been proposed for the reaction of NAD+ with primary and secondary alcohols (Silverstein & Boyer, 1964; Dalziel & Dickinson, 1966a,b; Ainslie & Cleland, 1972).…”