Histidine-51 Facilitates the Conformational Change and Deprotonation of Horse Liver Alcohol Dehydrogenase–NAD+ Complexes

Abstract

Structural studies suggest that His-51 in wild-type horse liver alcohol dehydrogenase (ADH1E) facilitates proton transfer between the hydroxyl group of a ligand bound to the catalytic zinc and bulk solvent. Previous work showed that the Gln-51 substitution increases the pK values for pH dependencies for various enzymatic reactions by about 1.5 units, and stopped-flow studies on the binding of NAD+ and pyrazole suggested that the equilibrium position for the global conformational change of the enzyme–NAD+ complex became disfavored by 1000-fold. That conclusion needed confirmation. Here, X–ray crystallography of six ternary complexes of H51Q ADH with NAD+ and pyrazole, caprate, 2,2,2-trifluoroethanol, or 2,3,4,5,6-pentafluorobenzyl alcohol, and NADH and water or N-cyclohexylformamide shows that all complexes have the same closed conformation as wild-type ADH. Gln-51 fits in the region occupied by His-51 and does not affect the positions of bound coenzymes or inhibitors that coordinate to the catalytic zinc. Stopped-flow studies show that the rate-limiting formation of ternary complexes with H51Q ADH and NAD+ depends upon deprotonation of the ligands that bind to the catalytic zinc (pyrazole or 2,2,2-trifluoroethanol). Furthermore, the binding of NAD+ and trifluoroethanol monitored by fluorescence and proton release show that the H51Q substitution disfavors the conformational change by about 3-fold as compared to wild-type ADH. The pK values of the enzyme–NAD+ complexes with water and other zinc ligands increase because His-51 no longer facilitates the deprotonation. The roles of the zinc-water and His-51 in the mechanism are exposed.