Vetter Natasha D, Palmer David R J
Department of Chemistry, University of Saskatchewan , 110 Science Place, Saskatoon, SK, Canada S7N 5C9.
Biochemistry. 2017 Apr 11;56(14):2001-2009. doi: 10.1021/acs.biochem.7b00079. Epub 2017 Mar 29.
Glucose-6-phosphate 3-dehydrogenase (NtdC) is an NAD-dependent oxidoreductase encoded in the NTD operon of Bacillus subtilis. The oxidation of glucose 6-phosphate by NtdC is the first step in kanosamine biosynthesis. The product, 3-oxo-d-glucose 6-phosphate (3oG6P), has never been synthesized or isolated. The NtdC-catalyzed reaction is very slow at low and neutral pH, and its rate increases to a maximum near pH 9.5. However, under alkaline conditions, the product is not stable because of ring opening followed by deprotonation of the 1,3-dicarbonyl compound. The absorbance band due to this enolate at 310 nm overlaps with that of the other enzymatic product, NADH, complicating kinetic measurements. We report the deconvolution of the resulting spectra of the reaction to determine the rate constants and likely kinetic mechanism. In doing so, we were able to determine the extinction coefficient of the enolate of 3oG6P (23000 M cm), which allowed the measurement of the first-order rate constant (5.51 × 10 s) and activation energy (93 kJ mol) of nonenzymatic enolate formation. Using deuterium-labeled substrates, we show that hydride transfer from carbon 3 is partially rate-limiting in the enzymatic reaction, and deuterium substitution on carbon 2 has no significant effect on the enzymatic reaction but lowers the rate of deprotonation of 3oG6P 4-fold. These experiments clearly establish the regiochemistry of the reactions. Coupling of the NtdC reaction with the subsequent step in the pathway, NtdA-catalyzed glutamate-dependent amino transfer, has a small but significant effect on the rate of NAD reduction, consistent with these enzymes working together to process the unstable metabolite.
葡萄糖-6-磷酸3-脱氢酶（NtdC）是一种由枯草芽孢杆菌NTD操纵子编码的依赖NAD的氧化还原酶。NtdC催化葡萄糖6-磷酸的氧化是卡那胺生物合成的第一步。产物3-氧代-D-葡萄糖6-磷酸（3oG6P）从未被合成或分离出来。NtdC催化的反应在低pH和中性pH下非常缓慢，其速率在pH 9.5附近增加到最大值。然而，在碱性条件下，由于1,3-二羰基化合物的开环和去质子化，产物不稳定。该烯醇盐在310 nm处的吸收带与另一种酶促产物NADH的吸收带重叠，使动力学测量变得复杂。我们报告了反应所得光谱的去卷积，以确定速率常数和可能的动力学机制。通过这样做，我们能够确定3oG6P烯醇盐的消光系数（23000 M⁻¹cm⁻¹），这使得能够测量非酶促烯醇盐形成的一级速率常数（5.51×10⁻³ s⁻¹）和活化能（93 kJ mol⁻¹）。使用氘标记的底物，我们表明从碳3转移氢化物在酶促反应中部分限速，并在碳2上进行氘取代对酶促反应没有显著影响，但使3oG6P的去质子化速率降低4倍。这些实验清楚地确定了反应的区域化学。NtdC反应与途径中的后续步骤，即NtdA催化的依赖谷氨酸的氨基转移相偶联，对NAD还原速率有微小但显著的影响，这与这些酶共同作用处理不稳定代谢物一致。
