title: Substitutions of isoleucine residues at the adenine binding site activate horse liver alcohol dehydrogenase.
abstract: The contributions of isoleucine residues 224 and 269 of horse liver alcohol dehydrogenase to binding of the adenine moiety of NAD and to catalysis were studied by replacing Ile-224 with glycine (I224G) and Ile-269 with serine (I269S). The kinetic mechanisms of wild-type and both mutated liver enzymes were ordered. Affinities for several adenosine derivatives were decreased 5-50-fold by both substitutions. The I269S mutation differentially destabilized binding of the complete coenzyme, as affinities for NAD+ and NADH were decreased about 60-fold with the I224G enzyme and 350-fold for the I269S enzyme. The I269S substitution increased the rate constants for the conformational change that occurs when NAD+ binds. The maximum velocities for ethanol oxidation increased 7-fold with the I224G enzyme and 26-fold with the I269S enzyme due to the faster release of NADH. Hydride transfer limits the rate of oxidation of ethanol by the I269S enzyme. Inhibition constants for the substrate analogues, 2,2,2-trifluoroethanol and N-methylformamide, and catalytic efficiencies (V/Km) for ethanol and acetaldehyde were not changed by the mutations, indicating that binding of the adenosine moiety of the coenzyme is not necessarily coupled to the subsequent reaction of substrates.
authors.affiliation: Department of Biochemistry, University of Iowa, Iowa City 52242.
authors.forename: F
authors.initials: F
authors.lastname: Fan
authors.forename: B V
authors.initials: BV
authors.lastname: Plapp
mesh_terms: D000225:Adenine / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D006736:Horses; D007532:Isoleucine / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data
chemical_list: D003067:Coenzymes; D007532:Isoleucine; D000426:Alcohol Dehydrogenase; D000225:Adenine