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. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Biochemistry Année : 2008

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.

Résumé

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 degrees 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 beta-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].

Domaines

Biophysique

Dates et versions

hal-00273835 , version 1 (16-04-2008)

Identifiants

Citer

Claire Gondeau, Laurent Chaloin, Andrea Varga, Béatrice Roy, Perrine Lallemand, et al.. 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.. Biochemistry, 2008, 47 (11), pp.3462-73. ⟨10.1021/bi7023145⟩. ⟨hal-00273835⟩
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