1QV6

HORSE LIVER ALCOHOL DEHYDROGENASE HIS51GLN/LYS228ARG MUTANT COMPLEXED WITH NAD+ AND 2,4-DIFLUOROBENZYL ALCOHOL

Summary for 1QV6

• Entry DOI: 10.2210/pdb1qv6/pdb
• Related: 1HLD 1MG0 1QV7
• Descriptor: Alcohol dehydrogenase E chain, ZINC ION, NICOTINAMIDE-ADENINE-DINUCLEOTIDE, ... (6 entities in total)
• Functional Keywords: dehydrogenase, alcohol, nicotinamide coenzyme, 2, 4-difluorobenzyl alcohol, his51gln/lys228arg mutant, horse liver, oxidoreductase
• Biological source: Equus caballus (horse)
• Cellular location: Cytoplasm: P00327
• Total number of polymer chains: 2
• Total formula weight: 81737.44

Authors

Lebrun, L.A.,Park, D.-H.,Ramaswamy, S.,Plapp, B.V. (deposition date: 2003-08-26, release date: 2004-01-20, Last modification date: 2023-08-16)

Primary citation

LeBrun, L.A.,Park, D.-H.,Ramaswamy, S.,Plapp, B.V.
Participation of histidine-51 in catalysis by horse liver alcohol dehydrogenase.
Biochemistry, 43:3014-3026, 2004

PubMed Abstract: Histidine-51 in horse liver alcohol dehydrogenase (ADH) is part of a hydrogen-bonded system that appears to facilitate deprotonation of the hydroxyl group of water or alcohol ligated to the catalytic zinc. The contribution of His-51 to catalysis was studied by characterizing ADH with His-51 substituted with Gln (H51Q). The steady-state kinetic constants for ethanol oxidation and acetaldehyde reduction at pH 8 are similar for wild-type and H51Q enzymes. In contrast, the H51Q substitution significantly shifts the pH dependencies for steady-state and transient reactions and decreases by 11-fold the rate constant for the transient oxidation of ethanol at pH 8. Modest substrate deuterium isotope effects indicate that hydride transfer only partially limits the transient oxidation and turnover. Transient data show that the H51Q substitution significantly decreases the rate of isomerization of the enzyme-NAD(+) complex and becomes a limiting step for ethanol oxidation. Isomerization of the enzyme-NAD(+) complex is rate limiting for acetaldehyde reduction catalyzed by the wild-type enzyme, but release of alcohol is limiting for the H51Q enzyme. X-ray crystallography of doubly substituted His51Gln:Lys228Arg ADH complexed with NAD(+) and 2,3- or 2,4-difluorobenzyl alcohol shows that Gln-51 isosterically replaces histidine in interactions with the nicotinamide ribose of the coenzyme and that Arg-228 interacts with the adenosine monophosphate of the coenzyme without affecting the protein conformation. The difluorobenzyl alcohols bind in one conformation. His-51 participates in, but is not essential for, proton transfers in the mechanism.

PubMed: 15023053
DOI: 10.1021/bi036103m

Experimental method

X-RAY DIFFRACTION (1.8 Å)