Elementary steps in the reaction mechanism of the alpha-ketoglutarate dehydrogenase multienzyme complex from Escherichia coli: kinetics of succinylation and desuccinylation.
The kinetics of the succinylation and the desuccinylation of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli have been studied at 4 degrees C in 2 mM thiamin pyrophosphate, 2 mM MgCl2, and 20 mM potassium phosphate (pH 7.0) by steady-state and quenched-flow techniques. The initial steady-state velocity for the reaction of the complex is inhibited by high concentrations of alpha-ketoglutarate. The data are consistent either with cooperative interactions between two catalytic sites or with the existence of an alpha-ketoglutarate regulatory site. The time course of the succinylation by alpha-ketoglutarate of the unmodified complex or the complex in which a fraction of the alpha-ketoglutarate decarboxylase subunits (E1) has been inhibited with N-ethylmaleimide reveals a complex kinetic process. A mechanism consistent with the kinetic data is proposed in which some E1 subunits succinylate one lipoic acid per E1 and other E1 subunits succinylate two lipoic acids per E1. Furthermore, each succinylation reaction occurs via a two-step process with rate constants of 49 and 89 s-1 at saturating concentrations of alpha-ketoglutarate for the first and second steps, respectively. At long times, 13-16 mol of succinate binds per mol of unmodified complex. The stoichiometry of binding obtained with N-ethylmaleimide-treated complex is initially lower but approaches the same values as for the unmodified complex over the course of minutes. Coenzyme A removes the succinyl groups on the unmodified enzyme with a rate constant greater than or equal to 200 s-1. The results obtained suggest a limited accessibility between sites on the complex.
D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008956:Models, Chemical; D013386:Succinates / Q000378:metabolism*
D013386:Succinates; D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex; D005033:Ethylmaleimide