McPhail Don, Cheung Man-Kim, Brown Judith, Shepherdson Margaret
School of Life Sciences, University of the West of England, Coldharbour Lane, Bristol, BS16 1QY, UK.
Arch Microbiol. 2009 Jan;191(1):73-82. doi: 10.1007/s00203-008-0430-2. Epub 2008 Sep 20.
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.
在研究嗜放射栖热菌中的嘧啶合成途径时，观察到两例异常行为。其一，针对氨甲酰磷酸合成酶的两种检测方法得到了截然不同的结果。两种方法均依赖于底物碳酸氢盐中¹⁴C的固定以产生放射性产物。在偶联检测中，该酶的氨甲酰磷酸产物在天冬氨酸和天冬氨酸转氨甲酰酶存在的情况下转化为氨甲酰天冬氨酸。在直接检测中，反应混合物中省略了天冬氨酸，氨甲酰磷酸转化为尿素。结果发现，直接检测中的放射性计数约为偶联检测中测量值的5%。第二个异常现象是，两种检测混合物中均省略谷氨酰胺对放射性碳的固定没有显著影响。这些结果表明，天冬氨酸氨基氮可能是通过底物通道途径合成谷氨酰胺的氮源，该途径将谷氨酰胺传递给氨甲酰磷酸合成酶，并且外部添加的谷氨酰胺无法进入其在酶上的结合位点。
