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].
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