Substituent Effects on the Ionization Step Regulating Desorption and Catalytic Oxidation of Alcohols Bound to Liver Alcohol Dehydrogenase

Abstract

1. Transient-state kinetic methods, based on the use of pyrazole as a displacing reagent and reporter ligand, have been applied to examine the pH dependence of rate and equilibrium constants for 2-chloroethanol and 2,2-dichloroethanol binding to the binary complex formed between liver alcohol dehydrogenase and NAD' .2. The apparent affinity of the enzyme . NAD' complex for the examined alcohols is dependent on two proton dissociation equilibria. One of these equilibria affects the rate of alcohol association to the binary complex with a ligand-independent pKa value of 7.6, attributable to ionization of zinc-bound water in the enzyme. NAD' complex. The second proton dissociation equilibrium regulates the rate of alcohol desorption from the enzyme . NAD' . alcohol complex and exhibits a pK, value of 5.4 and 4.5, respectively, with 2-chloroethanol and 2,2'-dichloroethanol as the ligand. Steady-state kinetic data are reported which indicate that the pK,-5.4-equilibrium controls also the apparent rate of enzymic hydride transfer from 2-chloroethanol to NAD '. 3. These results lend strong support to the mechanism of enzyme action proposed by Kvassman andPettersson according to which the enzyme . NAD' . alcohol complex participates in an obligatory proton dissociation step which regulates both desorption and catalytic oxidation of the alcohol ligand. The corresponding pKa value is shown to be lineary dependent on the pK, of the free alcohol ligand with a regression coefficient (Bronstedt a value) of about 0.6. The latter observation provides direct evidence that the effect of pH on the reactivity of the enzyme . NAD' . alcohol complex reflects an alcohol/alcoholate ion equilibration of the enzyme-bound substrate. 4. The particular mode of binding and properties of the active-site zinc ion in liver alcohol dehydrogenase suggest that the catalytic function of the metal ion can be related primarily to facilitation of the process of alcohol oxidation through facilitation of the alcoholate ion formation step now established to precede hydride transfer to NAD' Liver alcohol dehydrogenase catalyzes the oxidation of alcohols by NAD'. The enzyme operates by an effectively ordered ternary-complex mechanism (Scheme l), coenzyme binding preceding binding of the alcohol substrate [l]. Investigations of the effect of pH on the catalytic steps of alcohol binding [2,3] and ternary-complex interconversion [4,5] have shown that the enzyme . NAD' . alcohol complex participates in a proton dissociation equilibrium which regulates the reactivity of this reaction intermediate. Alcohol desorption occurs exclusively from the protonated form of the ternary complex, while hydride transfer from the alcohol substrate to NAD' requires the unprotonated form of the complex.It has been suggested in previous reports from our laboratory [3,5] that the effect of pH on the reactivity of the enzyme . NAD+ . alcohol complex derives from an alcohol/ alcoholate ion equilibration of the enzyme-bound substrate (Scheme 2). Determinations of the corresponding ac...

Participation of Histidine-51 in Catalysis by Horse Liver Alcohol Dehydrogenase,

“…The pK values of free water, ethanol, 2-chloroethanol, and 2,2,2-trifluoroethanol of 15.4, 15.9, 14.3, and 12.4, respectively, are decreased in the ternary complexes with the wildtype enzyme and NAD + to 7.6, 6.4, 5.5, and 4.3 (39). The systematic depression of pK values is probably due to ligation of the alcohol to the catalytic zinc adjacent to the positive charge of the nicotinamide ring.…”

Sorbitol dehydrogenase is a zinc enzyme.

“…The nature of the protein ligands (Table III) is important in providing a substrate-interacting metal centre with the necessary properties (cf. Kvassman et al, 1981;Dietrich and Zeppezauer, 1982). In alcohol dehydrogenase, the ligands are one histidine and two cysteine residues.…”

On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase

“…Kvassman et al [15] have established a linear free-energy relationship for the dissociation constant of the liver ADH ternary complex (K d ) and pK a of the free alcohol. A correlation of pK d versus alcohol pK a yielded a Brønsted α value of 0.61.…”

“…The proposed alkoxide is thought to be stabilized and precisely positioned for catalysis through both a hydrogen bond with the protonated or unprotonated side chain of Lys-295 and hydrogen bonds with the side chains of Asn-191 and Asn-300, which are the components of the oxyanion hole. With the assumption of an effect on depression of the pK a of the reactive hydroxyl, the function of this oxyanion hole in PsM2DH can be compared with the established role of Zn 2+ in medium-chain ADHs [7,8,15]. The active-site triad of Lys-295, Asn-191 and Asn-300 facilitate hydride transfer to C4 of NAD + by a combination of propinquity effects which enable efficient Brønsted catalysis by the wellaimed side chain of Lys-295 and support C-H bond cleavage in the substrate.…”