Phospholipids chiral at phosphorus. Steric course of the reactions catalyzed by phosphatidylserine synthase from Escherichia coli and yeast.
Phospholipids chiral at phosphorus. Steric course of the reactions catalyzed by phosphatidylserine synthase from Escherichia coli and yeast.
复制标题
DOI：
10.1021/bi00387a042
复制
发表时间：
1987-06
期刊：
影响因子：
2.9
通讯作者：
C. Raetz;G. Carman;W. Dowhan;R. Jiang;W. Waszkuć;W. Loffredo;M. Tsai
中科院分区：
文献类型：
--
作者：
C. Raetz;G. Carman;W. Dowhan;R. Jiang;W. Waszkuć;W. Loffredo;M. Tsai
通过以下程序阐明了由大肠杆菌和酵母的磷脂酰丝氨酸（PS）合酶催化的反应的空间过程。 1,2-二棕榈酰基-sn-甘油-3-[17O,18O]磷酸乙醇胺 ([17O,18O]DPPE) 的 RP 和 SP 异构体是通过对之前的程序稍加修改来合成的 [Bruzik, K., & Tsai, M.-D. (1984) J. Am。化学。苏克。 106, 747-754] 并通过与磷脂酶 D 一起孵育分别转化为 (RP)- 和 (SP)-1,2-二棕榈酰-sn-甘油-3-[16O,17O,18O]磷酸 ([16O,17O18O]DPPA)。 [16O,17O,18O]DPPA 与胞苷缩合 吡啶中的 5'-单磷酸吗啉酯提供 PS 合酶所需的底物，[17O,18O]胞苷 5'-二磷酸-1,2-二棕榈酰-sn-甘油 ([17O,18O]CDP-DPG)，作为几种同位素和构型异构体的混合物。将 [17O,18O]CDP-DPG 与 L-丝氨酸、PS 合酶（将 [17O,18O]CDP-DPG 转化为磷脂酰丝氨酸）和 PS 脱羧酶（催化磷脂酰丝氨酸脱羧）的混合物一起孵育，得到 [17O,18O]DPPE。起始[17O,18O]DPPE和产物的构型和同位素富集在DPPE三甲基硅烷化后通过31P NMR进行分析。结果表明，大肠杆菌 PS 合酶的反应在磷上保留构型进行，这表明涉及磷脂酰酶中间体的两步机制，而酵母 PS 合酶通过构型反转催化反应，这表明单置换机制。这些结果为大肠杆菌酶提出的乒乓机制和酵母酶提出的顺序 Bi-Bi 机制提供了强有力的支持，两者都基于先前的同位素交换实验。
The steric courses of the reactions catalyzed by phosphatidylserine (PS) synthase from Escherichia coli and yeast were elucidated by the following procedure. RP and SP isomers of 1,2-dipalmitoyl-sn-glycero-3-[17O,18O]phosphoethanolamine ([17O,18O]DPPE) were synthesized with slight modification of the previous procedure [Bruzik, K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754] and converted to (RP)- and (SP)-1,2-dipalmitoyl-sn-glycero-3-[16O,17O,18O]phosphoric acid ([16O,17O18O]DPPA), respectively, by incubating with phospholipase D. Condensation of [16O,17O,18O]DPPA with cytidine 5'-monophosphomorpholidate in pyridine gave the desired substrate for PS synthase, [17O,18O]cytidine 5'-diphospho-1,2-dipalmitoyl-sn-glycerol ([17O,18O]CDP-DPG), as a mixture of several isotopic and configurational isomers. Incubation of [17O,18O]CDP-DPG with a mixture of L-serine, PS synthase (which converted [17O,18O]CDP-DPG to phosphatidylserine), and PS decarboxylase (which catalyzes decarboxylation of phosphatidylserine) gave [17O,18O]DPPE. The configuration and isotopic enrichments of the starting [17O,18O]DPPE and the product were analyzed by 31P NMR following trimethylsilylation of the DPPE. The results indicate that the reaction of E. coli PS synthase proceeds with retention of configuration at phosphorus, which suggests a two-step mechanism involving a phosphatidyl-enzyme intermediate, while the yeast PS synthase catalyzes the reaction with inversion of configuration, which suggests a single-displacement mechanism. Such results lend strong support to the ping-pong mechanism proposed for the E. coli enzyme and the sequential Bi-Bi mechanism proposed for the yeast enzyme, both based on previous isotopic exchange experiments.