title: Mechanism of binding of horse liver alcohol dehydrogenase and nicotinamide adenine dinucleotide.
abstract: The binding of NAD+ to liver alcohol dehydrogenase was studied by stopped-flow techniques in the pH range from 6.1 to 10.9 at 25 degrees C. Varying the concentrations of NAD+ and a substrate analogue used to trap the enzyme-NAD+ complex gave saturation kinetics. The same maximum rate constants were obtained with or without the trapping agent and by following the reaction with protein fluorescence or absorbance of a ternary complex. The data fit a mechanism with diffusion-controlled association of enzyme and NAD+, followed by an isomerization with a forward rate constant of 500 s-1 at pH 8: E E-NAD+ *E-NAD+. The isomerization may be related to the conformational change determined by X-ray crystallography of free enzyme and enzyme-coenzyme complexes. Overall bimolecular rate constants for NAD+ binding show a bell-shaped pH dependence with apparent pK values at 6.9 and 9.0. Acetimidylation of epsilon-amino groups shifts the upper pK to a value of 11 or higher, suggesting that Lys-228 is responsible for the pK of 9.0. Formation of the enzyme-imidazole complex abolishes the pK value of 6.9, suggesting that a hydrogen-bonded system extending from the zinc-bound water to His-51 is responsible for this pK value. The rates of isomerization of E-NAD+ and of pyrazole binding were maximal at pH below a pK of about 8, which is attributable to the hydrogen-bonded system. Acetimidylation of lysines or displacement of zinc-water with imidazole had little effect on the rate of isomerization of the E-NAD+ complex.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Iowa, Iowa City 52242.
authors.forename: V C
authors.initials: VC
authors.lastname: Sekhar
authors.forename: B V
authors.initials: BV
authors.lastname: Plapp
mesh_terms: D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000465:Algorithms; D000818:Animals; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D009243:NAD; D000426:Alcohol Dehydrogenase