title: 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.
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 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].
authors.affiliation: Centre d'tudes d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), UMR 5236, CNRS-Université Montpellier 1-Université Montpellier 2, Montpellier cedex 2, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Gondeau
authors.forename: Laurent
authors.initials: L
authors.lastname: Chaloin
authors.forename: Andrea
authors.initials: A
authors.lastname: Varga
authors.forename: Béatrice
authors.initials: B
authors.lastname: Roy
authors.forename: Perrine
authors.initials: P
authors.lastname: Lallemand
authors.forename: Christian
authors.initials: C
authors.lastname: Périgaud
authors.forename: Tom
authors.initials: T
authors.lastname: Barman
authors.forename: Mária
authors.initials: M
authors.lastname: Vas
authors.forename: Corinne
authors.initials: C
authors.lastname: Lionne
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002021:Buffers; D002384:Catalysis; D003080:Cold Temperature; D005988:Glyceric Acids / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D000432:Methanol; D008958:Models, Molecular; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D002021:Buffers; D005988:Glyceric Acids; D000244:Adenosine Diphosphate; C005156:3-phosphoglycerate; D010735:Phosphoglycerate Kinase; D000432:Methanol