Ref HAL: hal-00599278_v1
DOI: 10.1016/j.jmb.2011.04.048
Résumé: Substrate antagonism has been described for a variety of enzymes with more than one substrate and is characterized by a lowering of the affinity of one substrate in the presence of the other(s). 3-Phosphoglycerate kinase (PGK) catalyzes phosphotransfer from 1,3-bisphosphoglycerate (bPG) to ADP to give 3-phosphoglycerate (PG) and ATP, and is subject to substrate antagonism. Because of the instability of bPG, antagonism has only been described between PG and ATP or ADP. Here, we show that antagonism also occurs between bPG and ADP. Using the stopped-flow method, we show that the dissociation constant for one substrate increases in the presence of the other, and that this decrease in affinity is mainly due to an increase in the dissociation rate constant. As a consequence, there is an increase in the overall interaction kinetics. Interestingly, in the presence of the mirror image of natural D-ADP, L-ADP (a good substrate for PGK), antagonism is absent. Using rapid-quench-flow, we studied the kinetics of ATP formation. The time courses present the following: (1) a lag with L-ADP, but not with D-ADP, the kinetics of which were similar to the interaction kinetics measured by stopped-flow; (2) a burst that is directed by the phosphotransfer; and (3) a steady-state that is rate limited by the release of product kinetics. Structural explanations for these results are proposed by analyzing the crystallographic structure of the fully closed conformation of PGK in complex with L-ADP, PG, and the transition-state analogue AlF4 − compared to previously determined structures.

Ref HAL: hal-00566611_v1
DOI: 10.1016/j.tetlet2010.12.105
Résumé: We report on the first solution synthesis of lamivudine 5'-diphosphate in both high yield and purity. Efficient synthesis of lamivudine 5'-monophosphate was obtained through lamivudine H-phosphonate oxydation by (-)-(8,8-dichlorocamphorylsulfonyl)oxaridine. Diphosphorylation was performed by nucleophilic substitution of the phosphorimidazolate derivative of lamivudine HPLC coupled with UV or MS detection was found to be an invaluable tool for the follow-up of phosphorylation reactions.

Ref HAL: hal-00533916_v1

Ref HAL: hal-00533906_v1

Ref HAL: hal-00402840_v1

Ref HAL: hal-00383197_v1
DOI: 10.1021/ac802443j

Ref HAL: hal-00257625_v1
PMID 18096512
DOI: 10.1016/j.bbrc.2007.12.061
Résumé: l-Nucleoside-analogues, mirror images of the natural d-nucleosides, are a new class of antiviral and anticancer agents. In the cell they have to be phosphorylated to pharmacologically active triphosphate forms, the last step seems to involve human 3-phosphoglycerate kinase (hPGK). Here we present a steady state kinetic and biophysical study of the interaction of the model compound l-MgADP with hPGK. l-MgADP is a good substrate with k(cat) and K(m) values of 685s(-1) and 0.27mM, respectively. Double inhibition studies suggest that l-MgADP binds to the specific adenosine-binding site and protects the conformation of hPGK molecule against heat denaturation, as detected by microcalorimetry. Structural details of the interaction in the enzyme active site are different for the d- and l-enantiomers (e.g. the effect of Mg(2+)), but these differences do not prevent the occurrence of the catalytic cycle, which is accompanied by the hinge-bending domain closure, as indicated by SAXS measurements.