CHARACTERIZATION OF THE REACTION OF L-SERINE AND INDOLE WITH ESCHERICHIA-COLI TRYPTOPHAN SYNTHASE VIA RAPID-SCANNING ULTRAVIOLET VISIBLE SPECTROSCOPY
CHARACTERIZATION OF THE REACTION OF L-SERINE AND INDOLE WITH ESCHERICHIA-COLI TRYPTOPHAN SYNTHASE VIA RAPID-SCANNING ULTRAVIOLET VISIBLE SPECTROSCOPY
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DOI：
10.1021/bi00357a032
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发表时间：
1986-05-06
期刊：
影响因子：
2.9
通讯作者：
DUNN, MF
中科院分区：
文献类型：
--
作者：
DREWE, WF;DUNN, MF
已经使用光谱范围300-550 nm的快速扫描停流(RSSF)紫外-可见光谱在不同的预混合条件下研究了吲哚和L-丝氨酸与大肠杆菌色氨酸合酶的α2β2复合物的预稳态反应。当α2.β2与吲哚和L-丝氨酸混合时，发现α2.β2的反应以三种可检测的弛豫形式发生(1/.tau.1>1/.tau.2>1/.tau.3)，其速率常数与在与L-丝氨酸的部分反应中观察到的三种弛豫相同[Drewe，W.F.，Jr.和Dunn，M.F. (1985)生物化学24, 3977-3987]。发现由于用2H取代丝氨酸的α-1H而产生的动力学同位素效应与仅用丝氨酸的反应中观察到的效应相似。观察到的光谱变化和同位素效应表明，L-丝氨酸和PLP的醛亚胺以及源自该外部醛亚胺的第一醌型化合物是在τ1期间积累的瞬时物质。这些中间体在τ2和τ3期间向α-氨基丙烯酸酯希夫碱的转化限制了通过吲哚和α-氨基丙烯酸酯中间体之间的C-C键形成而产生的第二醌型物质(λmax 476nm)的形成速率。 α2.β2-丝氨酸混合物与吲哚的前稳态反应由四个弛豫组成(1/.tau.1* > 1/.tau.2* > 1/.tau.3* > 1/.tau.4*)。 1/.tau.1*光谱变化发生在混合死区时间内，并且是由于与吲哚相互作用时α2.β2-丝氨酸混合物的光谱扰动造成的，而476-nm物种的出现以1/.tau.2*、1/.tau.3*和1/.tau.4*为特征。这些实验证明，L-丝氨酸反应的τ1、τ2和τ3中发生的事件对于达到稳态是必须的，并且α2.β2与L-丝氨酸的反应产物对吲哚具有高度反应性。
The pre-steady-state reaction of indole and L-serine with the .alpha.2.beta.2 complex of Escherichia coli tryptophan synthase has been investigated under different premixing conditions with rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy for the spectral range 300-550 nm. When .alpha.2.beta.2 was mixed with indole and L-serine, the reaction of .alpha.2.beta.2 was found to occur in three detectable relaxations (1/.tau.1 > 1/.tau.2 > 1/ .tau.3) with rate constants identical with the three relaxations seen in the partial reaction with L-serine [Drewe, W. F., Jr., and Dunn, M. F. (1985) Biochemistry 24, 3977-3987]. Kinetic isotope effects due to substitution of 2H for the .alpha.-1H of serine were found to be similar to the effects observed in the reaction with serine only. The observed spectral changes and isotope effects indicate that the aldimine of L-serine and PLP and the first quinoid derived from this external aldimine are transient species that accumulate during .tau.1. Conversion of these intermediates to the .alpha.-aminoacrylate Schiff base during .tau.2 and .tau.3 limits the rate of formation of the second quinoidal species (.lambda.max 476 nm) generated via C-C bond formation between indole and the .alpha.-aminoacrylate intermediate. The pre-steady-state reaction of the .alpha.2.beta.2-serine mixture with indole is comprised of four relaxations (1/.tau.1* > 1/.tau.2* > 1/.tau.3* > 1/.tau.4*). The 1/.tau.1* spectral changes occur within the mixing dead time and result from the perturbation of the spectrum of the .alpha.2.beta.2-serine mixture upon interaction with indole, while the appearance of the 476-nm species is characterized by 1/.tau.2*, 1/.tau.3*, and 1/.tau.4*. These experiments demonstate that the events that occur in .tau.1, .tau.2, and .tau.3 of the L-serine reaction are obligatory for the attainment of the steady state and that the product of the reaction of .alpha.2.beta.2 with L-serine is highly reactive toward indole.