Osumi T, Ebisuno T, Nakano H, Katsuki H
J Biochem. 1975 Oct;78(4):763-72. doi: 10.1093/oxfordjournals.jbchem.a130965.
Using a cell-free extract of Rhodospirillum rubrum, studies were made of the condensation reaction between propionyl-CoA and glyoxylate. When [14C]propionate was incubated with the extract in the presence of glyoxylate, ATP, CoA, Mg2+, and Mn2+, radioactivity was incorporated into several compounds. Two of the main products were characterized as citramalate (CMA) and erythro-beta-methylmalate (erythro-MMA) on the basis of their behavior compared with authentic samples of CMA and erythro-MMA in the following three analyses: (i) paper chromatography using two solvent systems, (ii) radio-gas chromatography on their methyl esters, and (iii) chemical conversion to readily crystallizable derivatives, that is, citramalyl chloralide for CMA, and thymine for MMA. The CMA was thought to be of L(+)-form based on the results of optical resolution with brucine and also its susceptibility to L(+)-citramalate lyase of Clostridium tetanomorphum. When the reaction was carried out with lower concentrations of the enzyme, only MMA was accumulated. However, when the reaction was allowed to proceed further after addition of higher concentrations of the enzyme and of excess semicarbazide to prevent further condensation, the amount of accumulated MMA was decreased and CMA was formed instead. Furthermore, the time course of MMA and CMA formation exhibited a pattern typical of a precursor-product relationship. From these results, it was concluded that MMA was formed by alpha-condensation between propionyl-CoA and glyoxylate, and that CMA was derived from MMA, possibly from its CoA derivative.
利用深红红螺菌的无细胞提取物，对丙酰辅酶A与乙醛酸之间的缩合反应进行了研究。当[14C]丙酸在乙醛酸、ATP、辅酶A、Mg2+和Mn2+存在的情况下与提取物一起孵育时，放射性被掺入到几种化合物中。根据在以下三种分析中与柠檬酸苹果酸（CMA）和赤藓糖-β-甲基苹果酸（赤藓糖-MMA）的真实样品相比的行为，将两种主要产物鉴定为柠檬酸苹果酸（CMA）和赤藓糖-β-甲基苹果酸（赤藓糖-MMA）：（i）使用两种溶剂系统的纸色谱法，（ii）对其甲酯进行放射性气相色谱法，以及（iii）化学转化为易于结晶的衍生物，即CMA的柠檬酸苹果酰氯醛和MMA的胸腺嘧啶。基于与马钱子碱的旋光拆分结果以及其对破伤风梭状芽孢杆菌的L(+)-柠檬酸苹果酸裂合酶的敏感性，认为CMA是L(+)-型。当用较低浓度的酶进行反应时，仅积累了MMA。然而，当在加入较高浓度的酶和过量的氨基脲以防止进一步缩合后让反应进一步进行时，积累的MMA量减少，取而代之的是形成了CMA。此外，MMA和CMA形成的时间进程呈现出典型的前体-产物关系模式。从这些结果得出结论，MMA是由丙酰辅酶A与乙醛酸之间的α-缩合形成的，并且CMA源自MMA，可能源自其辅酶A衍生物。
