Binding of nucleotides to guanylate kinase, p21<i><SUP>ras</SUP></i>, and nucleoside-diphosphate kinase studied by nano-electrospray mass spectrometry

作     者：Prinz, H Lavie, A Scheidig, AJ Spangenberg, O Konrad, M 

作者机构：Max Planck Inst Mol Physiol D-44227 Dortmund Germany Max Planck Inst Biophys Chem D-37070 Gottingen Germany 

出 版 物：《JOURNAL OF BIOLOGICAL CHEMISTRY》 (生物化学杂志)

年 卷 期：1999年第274卷第50期

页      面：35337-35342页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学] 

主　　题：结合部位 鸟嘌呤核苷酸类/代谢 鸟苷二磷酸/化学 鸟苷二磷酸/代谢 鸟苷四磷酸/代谢 鸟苷三磷酸/化学 鸟苷三磷酸/代谢 动力学 质谱分析法/方法 核苷二磷酸激酶/化学 核苷二磷酸激酶/代谢 核苷一磷酸激酶/化学 核苷一磷酸激酶/代谢 磷酸盐类/代谢 原癌基因蛋白质p21(ras)/化学 原癌基因蛋白质p21(ras)/代谢 重组蛋白质类/化学 重组蛋白质类/代谢 酿酒酵母菌/酶学 硫酸盐类/代谢 人类 

摘      要：The binding of nucleotides to three different nucleotide-binding proteins and to a control protein was studied by means of nano-electrospray mass spectrometry applied to aqueous nondenaturing solutions. The method leads to unambiguous identification of enzyme complexes with substrates and products but does not allow the determination of dissociation constants or even stoichiometries relevant to the binding in solution. For guanylate kinase (EC 2.7.4.8), the transfer of HPO3 between nucleotides was observed whenever a ternary complex with adenylate or guanylate nucleotides was formed. Guanosine 5 -tetraphosphate was generated after prolonged incubation with GDP or GTP, Mg2+ binding was considerably enhanced in functional high affinity complexes, such as observed between guanylate kinase and its bisubstrate inhibitor P-1-(5 -guanosyl)-P-5-(5 -adenosyl) pentaphosphate or with the tight nucleotide-binding protein p21(ras) and GDP. Nucleoside-diphosphate kinase (EC 2.7.4.6) itself was phosphorylated in accordance to its known ping-pong mechanism. All nucleotide-binding proteins were shown to bind sulfate (SO42-) with presumably high affinity and slow exchange rate. The binding of phosphate (PO43-) could be inferred indirectly from competition with SO42-.