Langley K E, Kennedy E P
J Bacteriol. 1978 Oct;136(1):85-95. doi: 10.1128/jb.136.1.85-95.1978.
The cytosine liponucleotides CDP-diglyceride and dCDP-diglyceride are key intermediates in phospholipid biosynthesis in Escherichia coli (C. R. H. Raetz and E. P. Kennedy, J. Biol. Chem. 248:1098--1105, 1973). The enzyme responsible for their synthesis, CTP:phosphatidic acid cytidylytransferase, was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin and was purified about 70-fold (relative to cell-free extracts) in the presence of detergent. In studies of the heat stability of the enzyme, activity decayed slowly at 63 degrees C. Initial velocity kinetic experiments suggested a sequential, rather than ping-pong, reaction mechanism; isotopic exchange reaction studies supported this conclusion and indicated that inorganic pyrophosphate is released before CDP-diglyceride in the reaction sequence. The enzyme utilized both CTP and dCTP as nucleotide substrate for the synthesis of CDP-diglyceride and dCDP-diglyceride, respectively. No distinction was observed between CTP and dCTP utilization in any of the purification, heat stability, and reaction mechanism studies. In addition, CTP and dCTP were competitive substrates for the partially purified enzyme. It therefore appears that a single enzyme catalyzes synthesis of both CDP-diglyceride and dCDP-diglyceride in E. coli. The enzyme also catalyzes a pyrophosphorolysis of CDP-diglyceride, i.e., the reverse of its physiologically important catalysis.
胞嘧啶脂核苷酸CDP - 甘油二酯和dCDP - 甘油二酯是大肠杆菌磷脂生物合成中的关键中间体（C.R.H.雷茨和E.P.肯尼迪，《生物化学杂志》248:1098 - 1105，1973）。负责其合成的酶，即CTP：磷脂酸胞苷转移酶，通过涉及去污剂洋地黄皂苷的差异提取程序从细胞膜中溶解出来，并在去污剂存在的情况下纯化了约70倍（相对于无细胞提取物）。在对该酶热稳定性的研究中，活性在63℃下缓慢衰减。初始速度动力学实验表明反应机制是顺序的，而非乒乓机制；同位素交换反应研究支持了这一结论，并表明在反应序列中无机焦磷酸在CDP - 甘油二酯之前释放。该酶分别利用CTP和dCTP作为核苷酸底物来合成CDP - 甘油二酯和dCDP - 甘油二酯。在任何纯化、热稳定性和反应机制研究中，均未观察到CTP和dCTP利用上的差异。此外，CTP和dCTP是部分纯化酶的竞争性底物。因此，在大肠杆菌中似乎是单一酶催化CDP - 甘油二酯和dCDP - 甘油二酯的合成。该酶还催化CDP - 甘油二酯的焦磷酸解反应，即其生理上重要催化作用的逆反应。
