Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, 90187 Umeå, Sweden.
Phytochemistry. 2012 Jul;79:39-45. doi: 10.1016/j.phytochem.2012.04.002. Epub 2012 May 1.
UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.
UDP-葡萄糖焦磷酸化酶（UGPase）是一种关键酶，负责生成 UDP-Glc，后者用于数百种糖基化反应，涉及将葡萄糖添加到各种化合物中。在这项研究中，对大麦 UGPase 的特性进行了研究，以了解其底物对蛋白质活性和四级结构的影响。其与 Glc-1-P 和 UTP 的 K(m)值分别为 0.33 和 0.25 mM。除了使用 Glc-1-P 作为底物外，该酶还具有相当的 Gal-1-P 活性；然而，Gal-1-P 的 K(m)值非常高（>10 mM），使得该反应在生理条件下不太可能发生。UGPase 的相对较宽的 pH 最佳范围为 6.5-8.5，无论反应方向如何。酶平衡常数为 0.4，表明对反应的 Glc-1-P 合成方向略有偏好。通过气相电泳迁移率大分子分析（GEMMA）研究了酶的四级结构，发现无论是在反应的哪个方向，单一或两种底物的添加都会影响酶的四级结构，导致酶从二聚体向单体（酶的活性形式）转变。酶的四级结构的底物诱导变化可能具有调节作用，以确保最大的活性。讨论了动力学和影响 UGPase 寡聚状态的因素。
