Substrate analogs as mechanistic probes for the bifunctional chorismate synthase from Neurospora crassa.
Substrate analogs as mechanistic probes for the bifunctional chorismate synthase from Neurospora crassa.
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底物类似物作为粗糙脉孢菌双功能分支酸合酶的机械探针。
DOI：
10.1021/bi00251a019
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Bartlett,PA
中科院分区：
文献类型：
--
作者：
Lauhon,CT;Bartlett,PA
摘要：已制备了EPSP(4-8)的类似物，并对它们作为粗脉孢子菌分支酸合成酶底物的活性进行了动力学表征。该酶似乎对烯醇醚侧链Z-位的取代和还原类似物的5-位的取代表现出严格的区别。乙醇基和(R)-乳基类似物4和(R)-5都是良好的底物，其中(R)-5具有比天然底物更高的V值。已发现包括EPSP在内的三种底物对该酶表现出显著的底物抑制作用，目前可以用一个非竞争模型来解释这一现象，该模型涉及形成一个催化不起作用的三元ESZ络合物。用EPSP在C-3处观察到对V为1.10±0.02的二次动力学同位素效应，表明C-O键断裂在饱和底物浓度下具有显著的动力学意义，在极限底物浓度下这种效应被严重抑制，D(V/K)=0.97±0.02。在C-6R的初级氚同位素效应中也发现了类似的效应，如先前观察到的[Balasubramanian，S.，Davies，G.M.，Coggins，J.R.，&Abell，C.(1991)J.Am化学。SoC。113,8945-8946]。降低类似物(/？)-5和(5)-6的C-6R的初级同位素效应显著大于EPSP。与EPSP相比，(R)-S的V和DV值更大，这证明在饱和条件下分支酸的释放是部分限速的。将该酶与还原的5-deazaFMN孵育没有导致任何可观察到的分支酸的形成，这与先前的结果一致，该结果表明还原的黄素参与了从EPSP[Ramjee，.首页--期刊主要分类--期刊细介绍--期刊题录与文摘-期刊详细文摘内容
Revised Manuscript Received September 8, 1994® abstract: Analogs of EPSP (4—8) have been prepared, and their activity as substrates for the chorismate synthase from Neurospora crassa has beencharacterized kinetically. The enzyme appears to show strict discrimination against substitution at the Z-position of the enol ether side chain as well as against substitution at the 5-position of the reduced analogs. Both the glycolyl and (R)-lactyl analogs 4 and (R)-5 are good substrates, with (R)-5 having a higher V value than the natural substrate. Three substrates, including EPSP, have been found to show significant substrate inhibition with this enzyme, which at present can be explained by a noncompetitive model involving formation of a catalytically incompetent, ternary ESz complex. A significant secondary kinetic isotope effect on V of 1.10±0.02 has beenobserved at C-3 with EPSP, indicating thatC—O bond cleavage is kinetically significant at saturating substrate concentration; this effect is severely depressed at limiting substrate, with D (V/K)=0.97±0.02. A similar effect is found for the primary deuterium isotope effect at C-6R, as observed previously [Balasubramanian, S., Davies, G. M., Coggins, J. R., & Abell, C.(1991) J. Am. Chem. Soc. 113, 8945—8946]. The primary isotope effects at C-6R with reduced analogs (/?)-5 and (5)-6 are significantly larger than those with EPSP. The larger values of V and DV for (R)-S, when compared to EPSP, are evidence that release of chorismate is partially rate-limiting under saturating conditions. Incubation of the enzyme with reduced 5-deazaFMN does not result in any observable formation of chorismate, consistent with previous results indicating that reduced flavin is chemically involved in the synthesis of chorismate from EPSP [Ramjee,. N., Balasubramanian, S., Abell, C., Coggins, J. R., Davies, G. M., Hawkes, TR, Lowe, D. J., &