Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis

Abstract

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5′-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2′-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme–NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ∼1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD+ and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.

Relaxation of nonproductive binding and increased rate of coenzyme release in an alcohol dehydrogenase increases turnover with a nonpreferred alcohol enantiomer

“…Furthermore, rate limitation of the catalyzed oxidation of ( S )‐ 1 has been proposed to be mainly coupled to the rate of NADH release from the enzyme, which is of the same magnitude as k cat . Since ADH‐A, like its related isoenzyme from horse liver, follows an ordered sequential Bi–Bi mechanism , the rate‐limiting release of the reduced nucleotide would be unchanged irrespective of which alcohol enantiomer has been oxidized, since the same ketone product would be produced in either case. Also, in an ordered mechanism the rate of nucleotide release only involves the binary E•NADH complex.…”

A Water Molecule Triggers Guest Exchange at a Mono‐Zinc Centre Confined in a Biomimetic Calixarene Pocket: a Model for Understanding Ligand Stability in Zn Proteins

“…Most interestingly, a mechanism involving a hemi labile ligand and double displacement and inversion of the pyramid at a Td Zn II centre has been proposed for substrate binding and product release in some enzymes. In Horse Liver Alcohol Deshydrogenase, [31] it has, for example, been shown that a glutamate residue (Figure 7) participates in a double‐displacement interchange mechanism, via a trigonal bipyramidal geometry. The residue displaces, in a first step, the water molecule bound to the metal ion, and in a second step, the substrate oxygen (an alcohol) displaces the glutamate residue to give rise to the structure observed in the ternary complexes (enzyme/cofactor NAD + /alcohol substrate).…”

“…In the case of hydrolytic zinc enzymes, the metal is most often bound to three amino acid residues, generally histidine (His), and a water molecule completes its coordination sphere in a tetrahedral environment [6] . This water molecule can then either be activated by deprotonation, thereby giving rise to a hydroxide ion at physiological pH (such as in carbonic anhydrases), [8] activated by a hydrogen‐bonded general base (e. g., in metallo‐peptidases), [9] or displaced by the substrate (e. g., alcohol dehydrogenases) [10] or an inhibitor [11] . This raises questions relative to the role of water molecules in substrate binding, product release, as well as in inhibitor efficiency when the latter presents a coordinating site.…”

Enzyme-Catalyzed Redox Reactions

“…Contudo, diferentes estudos apontam para um mecanismo muito mais complexo, podendo envolver um átomo de zinco pentacoordenado ou uma reação de duplo deslocamento com a participação de um resíduo glutamato. 58 Estudos realizados com a álcool desidrogenase da bactéria termófila Thermoanerobacter brockii (TbADH), por exemplo, apontaram para dois novos estados do zinco pentacoordenado, TR1 e TR2 (Esquema 14). 59 No primeiro estado (TR1) o Zn está coordenado a uma molécula de água e aos resíduos His59, Cys37, Asp150 e Glu60.…”