Malyl-CoA formation in the NAD-, CoASH-, and alpha-ketoglutarate dehydrogenase-dependent oxidation of 2-keto-4-hydroxyglutarate. Possible coupled role of this reaction with 2-keto-4-hydroxyglutarate aldolase activity in a pyruvate-catalyzed cyclic oxidation of glyoxylate.
The alpha-ketoglutarate dehydrogenase complex of either pig heart or Escherichia coli catalyzes a NAD- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate which is stereoselective toward the L-isomer of this hydroxyketo acid. L-Malyl-CoA is the product of the reaction; the evidence includes observing (a) a steady increase in absorbance at 230 nm during the oxidation of 2-keto-4-hydroxyglutarate, (b) a positive response of oxidation reaction mixtures to neutral hydroxylamine, (c) loss of the two foregoing results concomitant with release of thiol-reacting material and the formation of free malate when reaction mixtures are heated, (d) formation of a hydroxamate which has chromatographic mobilities identical to that of chemically synthesized malate hydroxamate, (e) enzymatic formation of a radioactive product from 14C-labeled 2-keto-4-hydroxyglutarate which co-migrates with chemically synthesized malyl-CoA, and (f) hydrolysis of the product by citrate synthase, an enzyme absolutely specific for citryl-CoA and L-malyl-CoA. A 1:1:1 stoichiometric relationship exists between the amount of 2-keto-4-hydroxyglutarate oxidized, NAD reduced, and malate (or malyl-CoA) formed. Results from studies in which either 14C-labeled 2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate is incubated with mixtures of purified enzymes or extracts of E. coli support the suggestion that the aldolase which preferentially catalyzes formation of L-2-keto-4-hydroxyglutarate from pyruvate plus glyoxylate in E. coli is coupled with the oxidative decarboxylation of this substrate, as reported here, and other enzymes in a multistep pyruvate-catalyzed cyclic oxidation of glyoxylate.
D000214:Acyl Coenzyme A / Q000096:biosynthesis*; D000818:Animals; D003065:Coenzyme A / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006038:Glyoxylates / Q000378:metabolism; D019811:Hydroxylamine; D006898:Hydroxylamines; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D013552:Swine
D000214:Acyl Coenzyme A; D006038:Glyoxylates; D006898:Hydroxylamines; D007656:Ketoglutaric Acids; D009243:NAD; C018989:2-keto-4-hydroxyglutarate; C042519:malyl-coenzyme A; D019811:Hydroxylamine; D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex; D007652:Oxo-Acid-Lyases; C021415:4-hydroxy-2-oxoglutarate aldolase; C031150:glyoxylic acid; D003065:Coenzyme A