Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase

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• 作者：Daniel Decker^a ; ¹ ; Meng Meng^a ; ¹ ; Agnieszka Gornicka^a ; Anders Hofer^b ; Malgorzata Wilczynska^b ; Leszek A. Kleczkowski^a ; ^{Leszek.kleczkowski@plantphys.umu.se}
• 关键词：aa ; amino acids ; AGPase ; ADP-Glc pyrophosphorylase ; BSA ; bovine serum albumin ; Gal ; galactose ; GALT ; Gal-1-P uridyltransferase ; GDH ; Glc-6-P dehydrogenase ; Glc ; glucose ; PGM ; phosphoglucomutase ; PPi ; pyrophosphate ; SuSy ; sucrose synthase ; UAGPase ; UDP-N-a
• 刊名：Phytochemistry
• 出版年：2012
• 期刊代码：85_00319422
• 类别：ch
• 出版时间：July, 2012
• 卷：79
• 期：Complete
• 页码：39-45
• 文件大小：633 K

摘要

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.