Fuller J Q, Leadlay P F
Biochem J. 1983 Sep 1;213(3):643-50. doi: 10.1042/bj2130643.
The reaction catalysed by methylmalonyl-CoA epimerase from Propionibacterium shermanii was studied in tritiated water, in the direction with (2R)-methylmalonyl-CoA as substrate, under 'irreversible' conditions. After partial reaction, even when most of the substrate had been converted into product (isolated as propionyl-CoA) essentially no solvent tritium appeared in residual (2R)-methylmalonyl-CoA. The product, however, did contain tritium, and the specific radioactivity of the (2S)-epimer was deduced to be 0.33 times that of the solvent. These results provide further support for the mechanism proposed for the epimerase-catalysed reaction in the accompanying paper [Leadlay & Fuller (1983) Biochem. J. 213, 635-642], in which two enzyme bases act respectively as proton donor and acceptor. The observed low discrimination against solvent tritium entering the product can be accounted for by a mechanism in which the release of product is slow, and the re-protonation step on the enzyme is reversible, without leading to isotopic exchange with the solvent.
在氚化水中，以（2R）-甲基丙二酰辅酶A为底物，在“不可逆”条件下，研究了谢氏丙酸杆菌甲基丙二酰辅酶A差向异构酶催化的反应。部分反应后，即使大部分底物已转化为产物（分离为丙酰辅酶A），残留的（2R）-甲基丙二酰辅酶A中基本上没有溶剂氚出现。然而，产物确实含有氚，且（2S）-差向异构体的比放射性推断为溶剂的0.33倍。这些结果为随附论文[Leadlay & Fuller (1983) Biochem. J. 213, 635 - 642]中提出的差向异构酶催化反应的机制提供了进一步支持，其中两个酶碱基分别作为质子供体和受体。观察到的对进入产物的溶剂氚的低区分度可以通过产物释放缓慢且酶上的再质子化步骤可逆的机制来解释，而不会导致与溶剂的同位素交换。
