Differences in the Transient Kinetics of the Binding of D-ADP and its Mirror Image L-ADP to Human 3-Phosphoglycerate Kinase Revealed by the Presence of 3-Phosphoglycerate

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• 作者：Claire Gondeau ; Laurent Chaloin ; Andrea Varga ; Bé ; atrice Roy ; Perrine Lallemand ; Christian Pé ; rigaud ; Tom Barman ; Má ; ria Vas ; Corinne Lionne
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：March 18, 2008
• 年：2008
• 卷：47
• 期：11
• 页码：3462 - 3473
• 全文大小：876K
• 年卷期：v.47,no.11(March 18, 2008)
• ISSN：1520-4995

文摘

L-Nucleosides comprise a new class of antiviral and anticancer agents that are converted in vivo by a cascade of kinases to pharmacologically active nucleoside triphosphates. The last step of the cascade may be catalyzed by 3-phosphoglycerate kinase (PGK), an enzyme that has low specificity for nucleoside diphosphate (NDP): NDP + 1,3-bisphosphoglycerate ↔ NTP + 3-phosphoglycerate. Here we compared the kinetics of the formation of the complexes of human PGK with D- and its mirror image L-ADP and the effect of 3-phosphoglycerate (PG) on these by exploiting the fluorescence signal of PGK that occurs upon its interaction with nucleotide substrate. Two types of experiment were carried out: equilibrium (estimation of dissociation constants) and stopped-flow (transient kinetics of the interactions). We show that under our experimental conditions (buffer containing 30% methanol, 4 °C) PGK binds D- and L-ADP with similar kinetics. However, whereas PG increased the dissociation rate constant for D-ADP by a factor of 8—which is a kinetic explanation for “substrate antagonism”—PG had little effect on this constant for L-ADP. We explain this difference by a molecular modeling study that showed that the β-phosphates of D- and L-ADP have different orientations when bound to the active site of human PGK. The difference is unexpected because L-ADP is almost as catalytically competent as D-ADP [Varga, A. et al. (2008) Biochem. Biophys. Res. Commun. 366, 994–1000].