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

Claire Gondeau, Laurent Chaloin, Andrea Varga, Béatrice Roy, Perrine Lallemand, Christian Périgaud, Tom Barman, Mária Vas, Corinne Lionne

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13 Citations (Scopus)

Abstract

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].

Original languageEnglish
Pages (from-to)3462-3473
Number of pages12
JournalBiochemistry
Volume47
Issue number11
DOIs
Publication statusPublished - Mar 18 2008

ASJC Scopus subject areas

  • Biochemistry

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