Lane A N, Kirschner K
Eur J Biochem. 1983 Jan 1;129(3):571-82. doi: 10.1111/j.1432-1033.1983.tb07087.x.
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.
采用停流技术研究了吲哚在色氨酸合酶活性位点与L-丝氨酸缩合的机制。单轮反应通过吲哚快速结合到预先形成的酶-L-丝氨酸复合物上发生，随后形成C-C键，L-色氨酸的α-碳负离子重新质子化，最终释放产物。还研究了吲哚C-3位的同位素取代、pH值以及吲哚丙醇磷酸的存在对这些过程的影响。吲哚的结合机制补充了已知的L-丝氨酸和L-色氨酸的结合机制，从而给出了详细的催化机制图景。它涉及两种有活性的酶-L-丝氨酸复合物，导致中心缩合过程存在分支途径。L-丝氨酸的脱水速率和L-色氨酸碳负离子的重新质子化速率可能受活性位点重排的限制。对吸收光谱、荧光光谱和圆二色光谱的分析，以及对用硼氢化物还原得到的立体异构体的已发表数据的分析表明，重排包括磷酸吡哆醛C-4'原子的重新定向。该机制为解释所有现有信息提供了详细框架，包括α亚基对β2亚基催化反应的激活作用。
