Contributions of valine-292 in the nicotinamide binding site of liver alcohol dehydrogenase and dynamics to catalysis

Biochemistry. 2001 Oct 23;40(42):12686-94. doi: 10.1021/bi011540r.


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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alcohol Dehydrogenase / chemistry
  • Alcohol Dehydrogenase / genetics
  • Alcohol Dehydrogenase / metabolism*
  • Animals
  • Binding Sites / genetics
  • Catalysis
  • Crystallography, X-Ray
  • Energy Transfer
  • Horses
  • Hydrogen-Ion Concentration
  • Kinetics
  • Liver / enzymology*
  • NAD / metabolism*
  • Protons
  • Substrate Specificity / genetics
  • Temperature
  • Thermodynamics
  • Valine / chemistry
  • Valine / genetics
  • Valine / metabolism*


  • Protons
  • NAD
  • Alcohol Dehydrogenase
  • Valine

Associated data

  • PDB/1JU9