Substitutions of a buried glutamate residue hinder the conformational change in horse liver alcohol dehydrogenase and yield a surprising complex with endogenous 3′-Dephosphocoenzyme A

Substitutions of Amino Acid Residues in the Substrate Binding Site of Horse Liver Alcohol Dehydrogenase Have Small Effects on the Structures but Significantly Affect Catalysis of Hydrogen Transfer

“…117 The results showed that conservative substitutions of the residues that are not in the active site have modest effects on activity and structure, and no evidence is found to support a significant role for fast, global dynamics in catalysis. In contrast, the G293A/P295T double substitution in a mobile loop 102 and the E267H 23 and V292S 78 substitutions hinder the slow conformational change that leads to the closed ternary complex observed for the wild-type enzyme and decrease the rate constant for hydride transfer from benzyl alcohol by 6−60-fold. These substitutions can affect the ability of the enzyme to preorganize the substrates for catalysis.…”

“…19 The enzymes were rechromatographed on DEAE-Sepharose to remove endogenous adenosine nucleotide, which was identified by X-ray crystallography in another study to be 3′-dephosphocoenzyme A that binds in the coenzyme binding site. 23 The A 280 /A 260 ratio for the wildtype enzyme is 1.45, but a value of 1.48 was observed for the F93W enzyme. The concentrations of purified proteins were determined by ultraviolet absorption.…”

“…Preparations of the S48T, F93A, S48T/F93A, and V203A enzymes were described previously. , The enzymes were purified to apparent homogeneity according to the published procedure . The enzymes were rechromatographed on DEAE-Sepharose to remove endogenous adenosine nucleotide, which was identified by X-ray crystallography in another study to be 3′-dephosphocoenzyme A that binds in the coenzyme binding site . The A 280 / A 260 ratio for the wild-type enzyme is 1.45, but a value of 1.48 was observed for the F93W enzyme.…”