The Binding of NADH to Succinic Semialdehyde Dehydrogenase

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Abstract
Succinic semialdehyde dehydrogenase from pig brain is a tetramer composed of identical subunits of MW 41,000. Fluorometric titrations conducted on samples of enzyme in pyrophosphate buffer (pH 8.4) reveal the presence of 2 NADH binding sites characterized by a Kd of 4 .mu.M. The unusual stoichiometry of binding of NADH, i.e., 2 mol NADH/enzyme tetramer, as demonstrated by emission anisotropy measurements, is not due to reversible association-dissociation of the oligomeric structure at pH 8.4. The results of the fluorometric titrations are consistent with a model of extreme negative cooperativity, i.e., the affinity of NADH for the enzyme is influenced by interactions between the protomers. The reaction catalyzed by succinic semialdehyde dehydrogenase is inhibited by the substrate succinic semialdehyde with a Ki of 0.09 mM which is 5-fold greater than the Km value. The binding of excess substrate to the catalytic site provides a direct and simple mechanism for regulation of the rate of product formation.