On the mechanism of α-oxoglutarate oxidation in Escherichia coli

Abstract

Cell-free extracts of the succinate-requiring mutant of E. coli, 309-1 R, and of its parent wild type E. coli W, were tested for their ability to catalyse reactions involved in the overall oxidation of [alpha]-oxoglutarate to succinate. Whereas extracts of the wild type catalysed the reduction of diphospyopyridine nucleotide in the presence of [alpha]-oxoglutarate, thiamin pyrophosphate and coenzyme A, extracts of the mutant did not. Addition of mutant extract to that of the wild type did not affect the latter''s ability to oxidize [alpha]-oxoglutarate; this shows that the inability of the mutant extract to effect this reaction was due not to the presence of an enzymic inhibitor, but to the lack of an enzymic component of the [alpha]-oxoglutarate-dehydrogenase system. Extracts of the wild type organism catalysed the anaerobic evolution of carbon dioxide from [alpha]-oxoglutarate in the presence of thiamin pyrophosphate and ferricyanide as electron acceptor. The rate of gas evolution was proportional to the amounts of extract added. Extracts of the mutant failed to effect this reaction. Extracts of the wild type organism in the presence of thiamin pyrophosphate catalysed the incorporation of isotope from [C14] bicarbonate into [alpha]-oxoglutarate, by isotopic exchange. Extracts of the mutant did not catalyse this reaction. Extracts of both types of organism contained succinic thiokinase, lipoic dehydrogenase and lipoic trans-succinylase a new assay procedure for the last-named enzyme is reported. These results show that the mutant lacked the enzymes catalysing the first step or first two steps in the overall oxidation of [alpha]-oxoglutarate. They also indicate that the formation of the succinic semialdehyde-thiamin pyrophosphate complex and carbon dioxide from [alpha]-oxoglutarate is effected by a discrete enzymic component of the [alpha]-oxoglutarate-dehydro-genase system. Undialysed cell-free extracts of the mutant, in the absence of added electron donors, catalysed the anaerobic reduction of [C14]malate to [C14]succinate: this reflects the ability of the mutant to grow anaerobically even in the absence of succinate.