Abstract
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.
Highlights
From the $Departments of Biochemistryand Molecular Biology and Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122 and the TDepartment of Biochemistry, University of California, Riverside, California92521
Human liver alcohol dehydrogenase isoenzymefsllfll encoded at ADH2, p&, P&, and p3p3, exhibitstrikingly and 8282, in which position47 in thecoenzyme binding different steady-state kinetic propertiefsor ethanol oxidation domain is an arginine or histidine, respectively, differwith NAD+ and for acetaldehyde reduction with NADH
Substitutions at position 47 stronglyinfluenceapparent DACA hydride studies of Arg-47 revealed that coenzyme steady-state kinetic parameters ( K, and Vmax)increase with substitution of a weaker base like histidine or a neutral residue like glutamine at position 47 [6]
Summary
TECH SF51 instrument, and traces were analyzed with HI-TECH software. Regressionswere analyzed with SAS (Cary, NC), evaluating. Estimates of k@CA and KgAcwAere determined by nonlinear regression of a first order hyperbolic equation, using 10 to 15 data points a t 5 to 7 DACA concentrations [18].To minimize exposure time of the enzymes below pH 6, pH-jump experiments were performed as described [18]. Human Alcohol Dehydrogenase Kinetics mixing 30 mM 4-methylpyrazole with enzyme, pre-equilibrated with 2 mM NAD+ These rates with P47H and P47R were not limiting across the pHrange studied (TableI). Dissociation rate constants (kzfD', s") were determined by mixing enzyme, preincubated with enough NAD' to bind 95% of the active sites, with excess DACA and varying NADH concentrations (Equation 3). Apparent NAD+ Association Rate Constants-Apparent NAD' association rate constants (k,",*", p"' s-') were determined by mixing enzyme with excess 1-butanol and DACA, and with varying NAD+ concentrations (Equation 5) [20]
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