The pH-Dependent Binding of NADH and Subsequent Enzyme Isomerization of Human Liver β3β3 Alcohol Dehydrogenase

• Published in: Biochemistry (Easton) 1999-05, Vol.38 (18), p.5829-5835
• Main Authors: Stone, Carol L, Jipping, Mary Beth, Owusu-Dekyi, Kwabena, Hurley, Thomas D, Li, Ting-Kai, Bosron, William F
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi982944v

Human class I β3β3 is one of the alcohol dehydrogenase dimers that catalyzes the reversible oxidation of ethanol. The β3 subunit has a Cys substitution for Arg-369 (β369C) in the coenzyme-binding site of the β1 subunit. Kinetic studies have demonstrated that this natural mutation in the coenzyme-binding site decreases affinity for NAD+ and NADH. Structural studies suggest that the enzyme isomerizes from an open to closed form with coenzyme binding. However, the extent to which this isomerization limits catalysis is not known. In this study, stopped-flow kinetics were used from pH 6 to 9 with recombinant β369C to evaluate rate-limiting steps in coenzyme association and catalysis. Association rates of NADH approached an apparent zero-order rate with increasing NADH concentrations at pH 7.5 (42 ± 1 s-1). This observation is consistent with an NADH-induced isomerization of the enzyme from an open to closed conformation. The pH dependence of apparent zero-order rate constants fit best a model in which a single ionization limits diminishing rates (pK a = 7.2 ± 0.1), and coincided with V max values for acetaldehyde reduction. This indicates that NADH-induced isomerization to a closed conformation may be rate-limiting for acetaldehyde reduction. The pH dependence of equilibrium NADH-binding constants fits best a model in which a single ionization leads to a loss in NADH affinity (pK a = 8.1 ± 0.2). Rate constants for isomerization from a closed to open conformation were also calculated, and these values coincided with V max for ethanol oxidation above pH 7.5. This suggests that NADH-induced isomerization of β369C from a closed to open conformation is rate-limiting for ethanol oxidation above pH 7.5.