Heavy atom isotope effects reveal a highly polarized transition state for chorismate mutase
Heavy atom isotope effects reveal a highly polarized transition state for chorismate mutase
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DOI：
10.1021/ja9841759
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发表时间：
1999-03-03
影响因子：
15
通讯作者：
Hilvert, D
中科院分区：
文献类型：
--
作者：
Gustin, DJ;Mattei, P;Hilvert, D
分支酸(1)转化为预苯酯(2)是由分支酸变位酶催化的。1酶反应在形式上是Claisen重排，通过主席式的过渡态进行，2但关于这种高能物种结构的更详细信息仍不清楚。动力学同位素效应(Kies)是确定过渡态几何结构的强大工具。例如，次级氚同位素效应表明，分支酸的非催化重排是通过不对称过渡态进行的，在该过渡态中，C(5)-O(5)键约有40%断裂，C(1)-C(9)键未被检测到形成。3这些结果与基于该反应的RHF/6-31*跃迁结构的计算一致4，并且对于脂肪族Claisen重排是典型的。5、6不幸的是，在来自大肠杆菌的双功能分支酸变位预苯酸脱氢酶催化的反应中没有观察到同位素效应。3本征同位素效应的抑制意味着在重排本身之前处于动力学上显著的过渡状态，可能涉及底物络合或蛋白质构象变化，3但这阻碍了对酶活性部位的化学反应的直接研究。粘度变化实验表明，扩散过渡态只部分限制了第二种分支酸变位酶，即来自枯草芽孢杆菌(BSCM)的单功能酶。7虽然BSCM和大肠杆菌分支酸变位酶有不同的三级折叠，8，9它们具有相似的活性和抑制谱，以及相似的功能化活性部位。9因为对粘度不敏感的高活性BSCM变体也
The conversion of chorismate (1) to prephenate (2) is catalyzed by the enzyme chorismate mutase. 1 The enzymatic reaction is formally a Claisen rearrangement, proceeding through a chairlike transition state, 2 but more detailed information on the structure of this high-energy species has remained elusive.Kinetic isotope effects (KIEs) are powerful tools in the determination of transition-state geometries. Secondary tritium isotope effects, for example, have shown that the uncatalyzed rearrangement of chorismate occurs via an asymmetric transition state in which the C (5)-O (5) bond is approximately 40% broken and the C (1)-C (9) bond is not detectably formed. 3 These results are in accord with calculations based on the RHF/6-31* transition structure for this reaction4 and are typical for aliphatic Claisen rearrangements. 5, 6 Unfortunately, no isotope effects were observed for the reaction catalyzed by the bifunctional chorismate mutaseprephenate dehydrogenase from Escherichia coli. 3 Suppression of the intrinsic isotope effect suggests a kinetically significant transition state prior to the rearrangement itself, presumably involving substrate complexation or a protein conformational change, 3 which nevertheless precludes direct study of the chemical reaction at the enzyme active site. Viscosity variation experiments have shown that diffusive transition states only partially limit a second chorismate mutase, the monofunctional enzyme from Bacillus subtilis (BsCM). 7 Although BsCM and E. coli chorismate mutases have different tertiary folds, 8, 9 they have comparable activities and inhibition profiles and similarly functionalized active sites. 9 Because highly active BsCM variants which are insensitive to viscosity are also