Department of Chemistry, University at Buffalo, The State University of New York , Buffalo, NY 14260, USA.
Biochemistry. 2012 Jul 3;51(26):5307-19. doi: 10.1021/bi300513r. Epub 2012 Jun 21.
As part of the non-mevalonate pathway for the biosynthesis of the isoprenoid precursor isopentenyl pyrophosphate, 1-deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase (DXR) catalyzes the conversion of DXP into 2-C-methyl-D-erythritol 4-phosphate (MEP) by consecutive isomerization and NADPH-dependent reduction reactions. Because this pathway is essential to many infectious organisms but is absent in humans, DXR is a target for drug discovery. In an attempt to characterize its kinetic mechanism and identify rate-limiting steps, we present the first complete transient kinetic investigation of DXR. Stopped-flow fluorescence measurements with Mycobacterium tuberculosis DXR (MtDXR) revealed that NADPH and MEP bind to the free enzyme and that the two bind together to generate a nonproductive ternary complex. Unlike the Escherichia coli orthologue, MtDXR exhibited a burst in the oxidation of NADPH during pre-steady-state reactions, indicating a partially rate-limiting step follows chemistry. By monitoring NADPH fluorescence during these experiments, the transient generation of MtDXR·NADPH·MEP was observed. Global kinetic analysis supports a model involving random substrate binding and ordered release of NADP(+) followed by MEP. The partially rate-limiting release of MEP occurs via two pathways--directly from the binary complex and indirectly via the MtDXR·NADPH·MEP complex--the partitioning being dependent on NADPH concentration. Previous mechanistic studies, including kinetic isotope effects and product inhibition, are discussed in light of this kinetic mechanism.
作为异戊烯焦磷酸非甲羟戊酸途径生物合成的异戊烯基焦磷酸（IPP）前体的一部分，1-脱氧-D-木酮糖-5-磷酸（DXP）还原异构酶（DXR）通过连续的异构化和 NADPH 依赖性还原反应，将 DXP 转化为 2-C-甲基-D-赤藓醇 4-磷酸（MEP）。由于该途径对许多传染性生物体是必不可少的，但在人类中不存在，因此 DXR 是药物发现的靶点。为了表征其动力学机制并确定限速步骤，我们首次对 DXR 进行了完整的瞬态动力学研究。使用结核分枝杆菌 DXR（MtDXR）的停流荧光测量表明，NADPH 和 MEP 与游离酶结合，并且两者结合在一起生成非生产性的三元复合物。与大肠杆菌同源物不同，MtDXR 在预稳态反应中显示 NADPH 的氧化发生爆发，表明随后的化学步骤存在部分限速步骤。通过在这些实验中监测 NADPH 荧光，可以观察到 MtDXR·NADPH·MEP 的瞬时生成。全局动力学分析支持一种涉及随机底物结合和按顺序释放 NADP(+) 然后释放 MEP 的模型。MEP 的部分限速释放通过两条途径发生 - 直接从二元复合物和间接通过 MtDXR·NADPH·MEP 复合物 - 分配取决于 NADPH 浓度。先前的机制研究，包括动力学同位素效应和产物抑制，都根据该动力学机制进行了讨论。
