GLYOXYLIC ACID CARBOLIGASE: AN ENZYME PRESENT IN GLYCOLATE-GROWN ESCHERICHIA COLI

Abstract

The principal reaction studied was found in cell-free extracts from Escherichia coli grown with a salts medium containing glycolic acid as a sole carbon source. The reaction was detected under anaerobic conditions with glyoxylic acid as the substrate. It was followed manometrically by the evolution of carbon dioxide or titrimetrically by neutralization of the hydrogen ion released. The stoichiometry indicated 1/2 mole of carbon dioxide released with the utilization of 1 mole of glyoxylic acid. A partial fractionation of the enzyme catalyzing the reaction was achieved with ammonium sulfate and ethanoL This permitted a definition of the cofactor requirements, namely thiamine pyrophosphate and Mg++ ions. A search for other products consistently revealed the presence of hydroxypyruvic acid as shown by chromatography of free acid products and 2,4-dinitrophenylhydrazone derivatives. Nonetheless, spectrophotometric experiments utilizing lactic dehydro-genase and D-glyceric dehydrogenase confirmed that the primary product was not hydroxypyruvic acid. The fortuitous presence in crude extracts of a diphosphepyridine nucleotide dependent dehydrogenase capable of reducing the primary product to glyceric acid helped to identify the primary product as tartronic semialdehyde. A tentative scheme was outlined, centeringonthe reaction studied in relation to known intermediary pathways of metabolism by which glycolate-grown E. coli could derive energy and build larger carbon skeletons.