title: Amino acid residues in the nicotinamide binding site contribute to catalysis by horse liver alcohol dehydrogenase.
abstract: Amino acid residues Thr-178, Val-203, and Val-292, which interact with the nicotinamide ring of the coenzyme bound to alcohol dehydrogenase (ADH), may facilitate hydride transfer and hydrogen tunneling by orientation and dynamic effects. The T178S, T178V, V203A, V292A, V292S, and V292T substitutions significantly alter the steady state and transient kinetics of the enzyme. The V292A, V292S, and V292T enzymes have decreased affinity for coenzyme (NAD+ by 30-50-fold and NADH by 35-75-fold) as compared to the wild-type enzyme. The substitutions in the nicotinamide binding site decrease the rate constant of hydride transfer for benzyl alcohol oxidation by 3-fold (for V292T ADH) to 16-fold (for V203A ADH). The modest effects suggest that catalysis does not depend critically on individual residues and that several residues in the nicotinamide binding site contribute to catalysis. The structures of the V292T ADH-NAD+-pyrazole and wild-type ADH-NAD+-4-iodopyrazole ternary complexes are very similar. Only subtle changes in the V292T enzyme cause the large changes in coenzyme binding and the small change in hydride transfer. In these complexes, one pyrazole nitrogen binds to the catalytic zinc, and the other nitrogen forms a partial covalent bond with C4 of the nicotinamide ring, which adopts a boat conformation that is postulated to be relevant for hydride transfer. The results provide an experimental basis for evaluating the contributions of dynamics to hydride transfer.
authors.affiliation: Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242, USA.
authors.forename: Jon K
authors.initials: JK
authors.lastname: Rubach
authors.forename: Bryce V
authors.initials: BV
authors.lastname: Plapp
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D018360:Crystallography, X-Ray / Q000379:methods; D006736:Horses; D006859:Hydrogen / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry; D009536:Niacinamide / Q000737:chemistry*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014633:Valine / Q000235:genetics
chemical_list: D000596:Amino Acids; D009243:NAD; D009536:Niacinamide; D006859:Hydrogen; D000426:Alcohol Dehydrogenase; D014633:Valine