Open AccessThe Binding of NADH to Succinic Semialdehyde Dehydrogenase
Author(s) -
BLANER William S.,
CHURCHICH Jorge E.
Publication year - 1980
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1980.tb04812.x
Subject(s) - tetramer , chemistry , dehydrogenase , cooperativity , dissociation constant , titration , stereochemistry , cooperative binding , substrate (aquarium) , enzyme , pyrophosphate , biochemistry , inorganic chemistry , biology , ecology , receptor
The enzyme succinic semialdehyde dehydrogenase from pig brain is a tetramer composed of identical subunits of molecular weight 41000. Fluorometric titrations conducted on samples of enzyme in pyrophosphate buffer (pH 8.4) reveal the presence of two NADH binding sites characterized by a dissociation constant of 4 μM. The unusual stoichiometry of binding of NADH, i.e. two moles 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 K i of 0.09 mM which is fivefold greater than the K m value. The binding of excess substrate to the catalytic site provides a direct and simple mechanism for regulation of the rate of product formation.