Alcohol dehydrogenase (ADH) catalyzes the reversible oxidation of alcohols to aldehydes by an ordered mechanism with the coenzyme NAD+ binding first, followed by binding of the alcohol, proton and hydride transfer, release of aldehyde, and dissociation of NADH. The oxygen of the alcohol is bound to a zinc in a tetrahedral coordination with two cysteine sulfurs and one histidine imidazole in the liver and yeast alcohol dehydrogenases.1,2 The zinc acts as a Lewis acid and stabilizes the intermediate alcoholate, which forms a low-barrier hydrogen bond with a nearby Ser or Thr hydroxyl group that is connected by a proton relay system to His-51 that acts as a base.3 In an analogue of the ternary product complex with NADH, the oxygen of a formamide also binds to the zinc.4 The catalytic zinc of the apo-enzyme (no bound coenzyme) of horse liver ADH binds a water in place of the alcohol. When the horse liver enzyme binds NAD+, the pK of a group (apparently the zinc-bound water) decreases from 9.5 to 7.3, and a proton is released (at pH 8.0) concurrently with a conformational change that closes up the active site and facilitates binding of substrate analogues.5, 6 The question arises, then, what is the mechanism for the exchange of the zinc-bound water (or hydroxide) with the substrate? An intermediate penta-coordinated zinc with adjacent oxygens from a water and the substrate for the enzyme-coenzyme-substrate complex has been proposed, but the evidence is not convincing; X-ray structures of relevant complexes have no such water. Nevertheless, such a penta-coordinated zinc could be a transient species, as the horse apo-enzyme forms stable penta-coordinated complexes with 2,2’-bipyridine and 1,10-phenanthroline. It is noteworthy that the zinc in the yeast apo-enzyme has an inverted configuration where the carboxylate of Glu-67 is in tetrahedral coordination with the two cysteines and the histidine.2, 7 Thus, an alternative mechanism could involve a double displacement where the carboxyl group of the nearby glutamate residue displaces the water, inverting the configuration of the zinc, and then the substrate oxygen displaces the carboxyl group. Such a mechanism is supported by structures that have alternative positions for the zinc in some ADH complexes and by studies that show catalytic efficiency for alcohol oxidation is decreased when the glutamate residue is substituted with neutral amino acids.8, 9 Computational studies of the horse enzyme also suggest that the glutamate can move to coordinate to the zinc.10 A role for Glu-68 (Glu-67 in yeast ADH) in the exchange of ligands on the catalytic zinc seems most reasonable.
(1) Plapp, B. V., and Ramaswamy, S. (2012) Atomic-resolution structures of horse liver alcohol dehydrogenase with NAD+ and fluoroalcohols define strained Michaelis complexes. Biochemistry 51, 4035-4048.
(2) Baskar Raj, S., Ramaswamy, S., and Plapp, B. V. (2014) Yeast alcohol dehydrogenase structure and catalysis. Biochemistry 53, 5791-5803.
(3) Sekhar, V. C., and Plapp, B. V. (1990) Rate constants for a mechanism including intermediates in the interconversion of ternary complexes by horse liver alcohol dehydrogenase. Biochemistry 29, 4289-4295.
(4) Venkataramaiah, T. H., and Plapp, B. V. (2003) Formamides mimic aldehydes and inhibit liver alcohol dehydrogenases and ethanol metabolism. J. Biol. Chem. 278, 36699-36706.
(5) Kovaleva, E. G., and Plapp, B. V. (2005) Deprotonation of the horse liver alcohol dehydrogenase-NAD+ complex controls formation of the ternary complexes. Biochemistry 44, 12797-12808.
(6) Plapp, B. V. (2010) Conformational changes and catalysis by alcohol dehydrogenase. Arch. Biochem. Biophys. 493, 3-12.
(7) Plapp, B. V., Charlier, H. A., Jr., and Ramaswamy, S. (2016) Mechanistic implications from structures of yeast alcohol dehydrogenase complexed with coenzyme and an alcohol. Arch. Biochem. Biophys. 591, 35-42.
(8) Ganzhorn, A. J., and Plapp, B. V. (1988) Carboxyl groups near the active site zinc contribute to catalysis in yeast alcohol dehydrogenase. J. Biol. Chem. 263, 5446-5454.
(9) Sanghani, P. C., Davis, W. I., Zhai, L., and Robinson, H. (2006) Structure-function relationships in human glutathione-dependent formaldehyde dehydrogenase. Role of Glu-67 and Arg-368 in the catalytic mechanism. Biochemistry 45, 4819-4830.
(10) Ryde, U. (1995) On the role of Glu-68 in alcohol dehydrogenase. Protein Sci. 4, 1124-1132.