The catalytic mechanism of tryptophan synthase from Escherichia coli. Kinetics of the reaction of indole with the enzyme--L-serine complexes.
The catalytic mechanism of tryptophan synthase from Escherichia coli. Kinetics of the reaction of indole with the enzyme--L-serine complexes.
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大肠杆菌色氨酸合酶的催化机制。
DOI：
10.1111/j.1432-1033.1983.tb07087.x
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
K. Kirschner
中科院分区：
文献类型：
--
作者：
A. Lane;K. Kirschner
采用停流技术研究了吲哚与L-丝氨酸在色氨酸合酶活性位点缩合的机理。单次转换是通过吲哚与预先形成的酶-L-丝氨酸复合物快速结合、随后形成CC键、L-色氨酸的α碳负离子的再质子化及其最终释放而发生的。还研究了吲哚 C-3 位同位素取代、pH 以及吲哚丙醇磷酸酯的存在对这些过程的影响。吲哚的结合机制补充了 L-丝氨酸和 L-色氨酸的已知结合机制，以详细描述催化机制。它调用两种活性酶——L-丝氨酸复合物，导致中央缩合过程的分支途径。 L-丝氨酸的脱水率和L-色氨酸碳负离子的再质子化率可能受到活性位点重排的限制。对吸收、荧光和圆二色性光谱的分析，以及通过硼氢化物还原获得的立体异构体的公开数据表明，重排包括磷酸吡哆醛 C-4' 原子的重新取向。该机制提供了一个详细的框架来解释所有可用的信息，包括α亚基对β2亚基催化的反应的激活作用。
The mechanism by which indole condenses with L-serine in the active site of tryptophan synthase was studied by the stopped-flow technique. The single turnover occurs by rapid binding of indole to the pre-formed enzyme--L-serine complex, followed by C--C bond formation, reprotonation of the alpha carbon carbanion of L-tryptophan, and its final release. The effects of isotopic substitution at C-3 of indole, of pH, and of the presence of indolepropanol phosphate on these processes were also studied. The mechanism of binding of indole complements the known mechanisms of binding of L-serine and L-tryptophan to give a detailed picture of the mechanism of catalysis. It invokes two competent species of enzyme--L-serine complexes, leading to a branched pathway for the central condensation process. The rates of dehydration of L-serine and reprotonation of the carbanion of L-tryptophan are probably limited by rearrangements at the active site. Analysis of absorption, fluorescence and circular dichroic spectra, as well as of published data on the stereoisomers obtained by reduction with borohydride, suggests that the rearrangement includes a reorientation of the pyridoxal phosphate C-4' atom. The mechanism provides a detailed framework for explaining all available information, including the activating effect of the alpha subunit on the reaction catalyzed by the beta 2 subunit.