Amino acid substitutions at position 47 of human beta 1 beta 1 and beta 2 beta 2 alcohol dehydrogenases affect hydride transfer and coenzyme dissociation rate constants.
Human liver alcohol dehydrogenase isoenzymes beta 1 beta 1 and beta 2 beta 2, in which position 47 in the coenzyme binding domain is an arginine or histidine, respectively, differ remarkably in steady-state kinetics. To understand which catalytic steps affect these kinetics, apparent coenzyme dissociation and association rate constants, and apparent 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) hydride transfer rate constants were obtained with stopped-flow kinetics. Enzymes containing site-specific mutations of Arg-47 in beta 1 beta 1 (beta 47R) to His (beta 2 beta 2 or beta 47H), Lys (beta 47K), or Gln (beta 47Q) were studied. Apparent coenzyme dissociation rate constants are greatly affected by substitutions at position 47, in which mutant enzymes with a weak base or a neutral residue at this position (beta 47H and beta 47Q) exhibit faster rate constants than beta 47R and beta 47K. Substitutions at position 47 have less effect on apparent coenzyme association rate constants. The kinetics of NADH association for beta 47H and beta 47Q are consistent with a two-step mechanism in which the bimolecular binding step is coupled to a unimolecular process. These findings indicate that the greater role of position 47 in coenzyme dissociation may occur after a coenzyme-induced isomerization. Substitutions at position 47 also strongly influence apparent DACA hydride transfer rate constants; hydride transfer is faster with mutant enzymes containing weak bases like histidine at this position. Steady-state kinetics, however, reveal that the rate-limiting step of both beta 47R and beta 47H for acetaldehyde reduction and for ethanol oxidation is coenzyme product dissociation. Thus, the different activities of beta 1 beta 1 and beta 2 beta 2 for ethanol oxidation and acetaldehyde reduction are caused primarily by different coenzyme dissociation rates.
Stone
C L
CL
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
Bosron
W F
WF
Dunn
M F
MF
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002848:Chromatography, DEAE-Cellulose; D002934:Cinnamates / Q000378:metabolism; D004795:Enzyme Stability; D006736:Horses; D006801:Humans; D007202:Indicators and Reagents; D007536:Isomerism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008962:Models, Theoretical; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D013997:Time Factors
D002934:Cinnamates; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D009243:NAD; D001120:Arginine; C018523:4-dimethylaminocinnamaldehyde; D000426:Alcohol Dehydrogenase