Divalent cation and pH dependent primary isotope effects in the enolase reaction.
Divalent cation and pH dependent primary isotope effects in the enolase reaction.
复制标题
烯醇酶反应中二价阳离子和 pH 依赖性主要同位素效应。
DOI：
10.1021/bi00741a028
复制
发表时间：
1973
期刊：
影响因子：
2.9
通讯作者：
E. Westhead
中科院分区：
文献类型：
--
作者：
T. Shen;E. Westhead
方法制备[-H]-2-磷酸甘油酸。将Tes或Pipes缓冲盐溶解于99.5%D20中以制备0.05 μ m溶液，然后在室温下调节至pD 6.4。通过公式pD= pH+ 0.4获得pD（Lumry等人，1951年）。在典型的制备中，将约IOOmg的β-烯醇丙酮酸-三环己基胺盐溶解在IOml的含有20 mM MgS 〇 4的上述缓冲溶液中。加入1 - 2 mg纯酵母烯醇化酶，并将混合物在30 ℃下孵育。使用的缩写是：β-烯醇丙酮酸、磷酸烯醇丙酮酸; Tes和Pipes缓冲液，分别是N-三（羟甲基）甲基-2-氨基乙磺酸和哌嗪-N，N ′-双（2-乙磺酸）单钠一水合物。在本文中，同位素取代的底物[2 H]-2-磷酸甘油酸仅在碳原子2处用氘取代氢。
MethodsPreparation of [-H]-2-Phosphoglyceric Acid. Tes or Pipes buffer salts were dissolved in 99.5% D20 to make 0.05 m solutions, then adjusted to pD 6.4 at room temperature. The pD is obtained through the formula, pD= pH+ 0.4 (Lumry et al., 1951). In a typical preparation, about 100 mg of P-enolpyruvate-tricyclohexylamine salt was dissolved in 10 ml of the above buffer solutions containing 20 mM MgS04. Between 1 and 2 mg of pure yeast enolase was added and the mixture was incubated at 30. The progress of the reaction1 Abbreviations used are: P-enolpyruvate, phosphoenolpyruvate; Tes and Pipes buffers, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid and piperazine-jV, jV'-bis (2-ethanesulfonic acid) mono-sodium monohydrate, respectively. Throughout this paper the isotopically substituted substrate,[2H]-2-phosphoglyceric acid, has deuterium substituted for hydrogen only at carbon atom 2.