The structures and functions of alcohol dehydrogenases (ADHs) have been intensively studied because of their roles in the metabolism of alcohols. ADH catalyzes transfer of a proton from an alcohol to solvent and of a hydride from an alcohol to NAD+ to form an aldehyde and NADH [1]. Biochemical studies of the mechanisms and substrate specificities of ADHs provide fundamental knowledge about catalysis and useful information for commercial applications, such as diagnostic reagents and production of alcohols. The present work investigates the enzymatic mechanism of Saccharomyces cerevisiae ADH1, with comparisons to horse liver ADH1E, when the two histidines that can participate directly in catalysis are substituted.

Three-dimensional structures and site-directed mutagenesis have facilitated studies of catalysis by yeast ADH1. X-ray crystallography and cryo-electron microscopy established structures for several forms of the yeast ADH1 that represent various steps in the general mechanism of alcohol dehydrogenases [[2], [3], [4]]. The structures show that histidine residues 44 and 48 are in positions of catalytic importance (Fig. 1). His-44 forms a hydrogen bond with O2A of the pyrophosphate of the NAD(H) [5]. His-44 was changed to an arginine residue in yeast ADH, which is present in many wild-type ADHs, such as horse liver ADH [6]. His-48 hydrogen bonds with the 2′-hydroxyl group of the nicotinamide ribose, and potentially acts in acid/base catalysis through the hydrogen bonded system connected to the oxygen of the substrate (see Fig. 1 for yeast ADH and Scheme 1 for horse liver ADH in Ref. [7]). In some ADHs, this residue is a tyrosine, threonine, or glutamate, and it was replaced with serine, glutamate, or glutamine residues in previous studies [5,[7], [8], [9], [10], [11]]. In the present study both histidines were replaced to make yeast H44R/H48Q ADH, which should form a productive structure, as shown in Fig. 1. The double substitution might be expected to make a pH independent enzyme, or to expose the contributions of other enzymatic groups to the pH dependences, such as the water or alcohol ligated to the catalytic zinc.

The roles of histidine residues in alcohol dehydrogenases and many other enzymes have been studied by chemical modification with diethyl pyrocarbonate, a toxic sterilizing reagent used to inactivate ribonucleases [12]. Although the reagent is relatively specific for histidine residues, its use to identify “essential” histidines needs to be evaluated and validated. Comparison of the reactions of the wild-type yeast ADH and variants with histidine residues substituted with residues that cannot react with diethyl pyrocarbonate provide a lesson in the interpretation of the modification studies.

A role for histidine residues in catalysis by yeast ADH was suggested because enzymatic reactions are pH dependent with pK values of 7–8, and the rate of inactivation by diethyl pyrocarbonate increased with pH with a pK value of 7.1 for free enzyme and a pK of 8.7 for the enzyme-NADH complex [[13], [14], [15], [16]]. It was suggested that one histidine residue was involved in binding the coenzyme or in acid/base catalysis, although the identity of the residue was not established, and a three dimensional structure was not known at the time. His-44 and His-48 can be substituted conservatively (H44R, H48E, H48Q, and H48S) without completely knocking out activity (but with modest changes in the kinetics of catalysis), and it remains to be determined if these two residues are the ones reacting with diethyl pyrocarbonate and if there are other “essential” histidine residues [6,7,9]. Of the remaining 8 histidine residues, 6 are exposed to solvent (sequence numbers 15, 51, 113, 138, 171, and 240). His-15 is near the active site, but it does not appear to be involved in activity because the H15R substitution has only small effects on activity [17]. The nitrogens of the imidazole group of His-66 are ligated to the catalytic zinc and hydrogen bonded to Asp-46, and His-121 is buried with close interactions with protein residues, so that both of these histidines are probably not readily accessible to chemical modification [2,4].