RESUMO
In studying the pyrimidine synthesising pathway in Deinococcus radiophilus two instances of anomalous behaviour were observed. One was the strikingly different results obtained for two types of assay for carbamoyl phosphate synthetase. Both depend on the fixation of 14C from the substrate bicarbonate to give radioactive products. In the coupled assay the carbamoyl phosphate product of the enzyme is converted to carbamoyl aspartate in the presence of aspartate and aspartate transcarbamoylase. In the direct assay aspartate is omitted from the reaction mixture and the carbamoyl phosphate is converted to urea. It was found that the radioactive counts in the direct assay were about 5% of those measured in the coupled assay. The second anomaly was that omission of glutamine from both assay mixtures had no significant effect on the fixation of radioactive carbon. These results suggested that aspartate amino-N could be the source of nitrogen for glutamine synthesis by a substrate-channelled pathway which delivered glutamine to carbamoyl phosphate synthetase, and that externally added glutamine could not access its binding site on the enzyme.
Assuntos
Deinococcus/química , Deinococcus/metabolismo , Pirimidinas/metabolismo , Aspartato Carbamoiltransferase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/química , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/isolamento & purificação , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Carbamoil-Fosfato/metabolismo , Deinococcus/enzimologia , Di-Hidro-Orotato Desidrogenase , Glutamina/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/isolamento & purificação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Especificidade por SubstratoRESUMO
Aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase) catalyse the first two steps unique to pyrimidine synthesis. In many bacteria they form non-covalently bonded complexes. There are two types of DHOase, type I and type II which share a common ancestry. Type I is the more ancient form and is present in the complexes. In recently evolved bacteria the DHOase is defective and its function has been replaced by a type II DHOase which is separate from the complex. Deinococcus radiophilus diverges early on the phylogenetic tree and so might be expected to have an active type I DHOase. Purification of the 500 kDa ATCase-DHOase complex, by conventional techniques, showed it to possess an active DHOase.