Participation of histidine-51 in catalysis by horse liver alcohol dehydrogenase.
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.
LeBrun
Laurie A
LA
Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242-1109, USA.
Park
Doo-Hong
DH
Ramaswamy
S
S
Plapp
Bryce V
BV
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D041961:Deuterium Exchange Measurement; D005973:Glutamine / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D006736:Horses; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry
D005973:Glutamine; D009243:NAD; D006639:Histidine; D000426:Alcohol Dehydrogenase