Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, United States.
Biochemistry. 2013 Feb 26;52(8):1456-65. doi: 10.1021/bi301593c. Epub 2013 Feb 14.
Human UDP-α-d-glucose 6-dehydrogenase (hUGDH) forms a hexamer that catalyzes the NAD(+)-dependent oxidation of UDP-α-d-glucose (UDG) to produce UDP-α-d-glucuronic acid. Mammalian UGDH displays hysteresis (observed as a lag in progress curves), indicating that the enzyme undergoes a slow transition from an inactive to an active state. Here we show that hUGDH is sensitive to product inhibition during the lag. The inhibition results in a systematic decrease in steady-state velocity and makes the lag appear to have a second-order dependence on enzyme concentration. Using transient-state kinetics, we confirm that the lag is in fact due to a substrate and cofactor-induced isomerization of the enzyme. We also show that the cofactor binds to the hUGDH:UDG complex with negative cooperativity. This suggests that the isomerization may be related to the formation of an asymmetric enzyme complex. We propose that the hysteresis in hUGDH is the consequence of a functional adaptation; by slowing the response of hUGDH to sudden increases in the flux of UDG, the other biochemical pathways that use this important metabolite (i.e., glycolysis) will have a competitive edge.
人源 UDP-α-d-葡萄糖 6-脱氢酶（hUGDH）形成六聚体，催化 NAD(+)依赖的 UDP-α-d-葡萄糖（UDG）氧化，生成 UDP-α-d-葡糖醛酸。哺乳动物 UGDH 表现出滞后（在进展曲线中观察到延迟），表明该酶经历从非活性状态到活性状态的缓慢转变。本文中，我们发现 hUGDH 在滞后过程中对产物抑制敏感。这种抑制导致稳态速度系统下降，使滞后表现出对酶浓度的二级依赖性。通过瞬态动力学，我们证实滞后实际上是由于酶的底物和辅因子诱导的异构化。我们还表明，辅因子与 hUGDH:UDG 复合物具有负协同结合。这表明这种异构化可能与不对称酶复合物的形成有关。我们提出 hUGDH 的滞后是功能适应的结果；通过减缓 hUGDH 对 UDG 通量突然增加的反应，其他使用这种重要代谢物（即糖酵解）的生化途径将具有竞争优势。
