title: Contributions of valine-292 in the nicotinamide binding site of liver alcohol dehydrogenase and dynamics to catalysis.
abstract: The participation of Val-292 in catalysis by alcohol dehydrogenase and the involvement of dynamics were investigated. Val-292 interacts with the nicotinamide ring of the bound coenzyme and may facilitate hydride transfer. The substitution of Val-292 with Ser (V292S) increases the dissociation constants for the coenzymes (NAD(+) by 50-fold, NADH by 75-fold) and the turnover numbers by 3-7-fold. The V292S enzyme crystallized in the presence of NAD(+) and 2,3,4,5,6-pentafluorobenzyl alcohol has an open conformation similar to the structure of the wild-type apo-enzyme, rather than the closed conformation observed for ternary complexes with wild-type enzyme. The V292S substitution perturbs the conformational equilibrium of the enzyme and decreases the kinetic complexity, which permits study of the hydride transfer step with steady-state kinetics. Eyring plots show that the DeltaH for the oxidation (V(1)) of the protio and deuterio benzyl alcohols is 13 kcal/mol and that the kinetic isotope effect of 4.1 is essentially temperature-independent. Eyring plots for the catalytic efficiency for reduction of benzaldehyde (V(2)/K(p)) with NADH or NADD are distinctly convex, being temperature-dependent from 5 to 25 degrees C and temperature-independent from 25 to 50 degrees C; the kinetic isotope effect of 3.2 for V(2)/K(p) is essentially independent of the temperature. The temperature dependencies and isotope effects for V(1) and V(2)/K(p) are not adequately explained by semiclassical transition state theory and are better explained by hydride transfer occurring through vibrationally assisted tunneling.
authors.affiliation: Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242, USA.
authors.forename: J K
authors.initials: JK
authors.lastname: Rubach
authors.forename: S
authors.initials: S
authors.lastname: Ramaswamy
authors.forename: B V
authors.initials: BV
authors.lastname: Plapp
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D018360:Crystallography, X-Ray; D004735:Energy Transfer; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D011522:Protons; D013379:Substrate Specificity / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D014633:Valine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011522:Protons; D009243:NAD; D000426:Alcohol Dehydrogenase; D014633:Valine