1JU9

HORSE LIVER ALCOHOL DEHYDROGENASE VAL292SER MUTANT

Summary for 1JU9

• Entry DOI: 10.2210/pdb1ju9/pdb
• Descriptor: ALCOHOL DEHYDROGENASE, ZINC ION, NICOTINAMIDE-ADENINE-DINUCLEOTIDE, ... (4 entities in total)
• Functional Keywords: dehydrogenase, alcohol, nicotinamide coenzyme, oxidoreductase, mutant
• Biological source: Equus caballus (horse)
• Cellular location: Cytoplasm: P00327
• Total number of polymer chains: 2
• Total formula weight: 81270.93

Authors

Rubach, J.K.,Ramaswamy, S.,Plapp, B.V. (deposition date: 2001-08-24, release date: 2001-09-19, Last modification date: 2023-08-16)

Primary citation

Rubach, J.K.,Ramaswamy, S.,Plapp, B.V.
Contributions of valine-292 in the nicotinamide binding site of liver alcohol dehydrogenase and dynamics to catalysis.
Biochemistry, 40:12686-12694, 2001

PubMed 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.

PubMed: 11601993
DOI: 10.1021/bi011540r

Experimental method

X-RAY DIFFRACTION (2 Å)