Human aldehyde dehydrogenase: coenzyme binding studies

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Abstract
The binding of NADH and NAD+ to the human liver cytoplasmic, E1, and mitochondrial, E2, isozymes at pH 7.0 and 25.degree.C was studied by the NADH fluorescence enhancement technique, the sedimentation technique, and steady-state kinetics. The binding of radiolabeled [14C]NADH and [14C]NAD+ to the E1 isozyme when measured by the sedimentation technique yielded linear Scatchard plots with a dissociation constant of 17.6 .mu.M for NADH and 21.4 .mu.M for NAD+ and a stoichiometry of ca. two coenzyme molecules bound per enzyme tetramer. The dissociation constant, 19.2 .mu.M, for NADH as competitive inhibitor was found from steady-state kinetics. With the mitochondrial E2 isozyme, the NADH fluorescence enhancement technique showed only one, high-affinity binding site (KD = 0.5 .mu.M). When the sedimentation technique and radiolabeled coenzymes were used, the binding studies showed nonlinear Scatchard plots. A minimum of two binding sites with lower affinity was indicated for NADH (KD = 3-6 .mu.M and KD = 25-30 .mu.M) and also for NAD+ (KD = 5-7 .mu.M and KD = 15-30 .mu.M). A fourth binding site with the lowest affinity (KD = 184 .mu.M for NADH and KD = 102 .mu.M for NAD+) was observed from the steady-state kinetics. The dissociation constant for NAD+, determined by the competition with NADH via fluorescence titration, was found to be 116 .mu.M. The number of binding sites found by the fluorescence titration (n = 1 for NADH) differs from that found by the sedimentation technique (n = 1.8 - 2.2 for NADH and n = 1.2 - 1.6 for NAD+). The binding characteristics of NADH and NAD+ with the human mitochondrial, E2, isozyme may be rationalized on the basis of negative cooperativity induced by the bound ligand.