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Interaction of human 3-phosphoglycerate kinase with L-ADP, the mirror image of D-ADP |
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| Authors: | Varga Andrea Szabó Judit Flachner Beáta Roy Béatrice Konarev Peter Svergun Dmitri Závodszky Péter Périgaud Christian Barman Tom Lionne Corinne Vas Mária |
| Affiliation: | a Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina Str. 29, P.O. Box 7, H-1518 Budapest, Hungary b Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS, Universités Montpellier 1 et 2, case courrier 1705, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier cedex 5, France c European Molecular Biology Laboratory, Hamburg Outstation, Notkestrasse 85, D-22603 Hamburg, Germany d Institute of Crystallography, Russian Academy of Sciences, Leninsky pr. 59, 117333 Moscow, Russia e Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé (CPBS), UMR 5236 CNRS, Universités Montpellier 1 et 2, Institut de Biologie, 4 bd Henri IV, CS69033, 34965 Montpellier cedex 2, France |
| Abstract: | 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 kcat and Km values of 685 s−1 and 0.27 mM, 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 Mg2+), 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. |
| Keywords: | AMP-PCP, β,γ-methylene-adenosine-5&prime triphosphate AMP-PNP, β,γ-imido-adenosine-5&prime triphosphate 1,3-bPG, 1,3-bisphosphoglycerate Pi, inorganic phosphate DSC, differential scanning calorimetry DTT, dithiothreitol GAP, smallcaps" >d-glyceraldehyde-3-phosphate GAPDH, smallcaps" >d-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) HIV, human immunodeficiency virus Nbs2, Ellman&rsquo s reagent, 5,5&prime -dithiobis-(2-nitrobenzoic acid) 3-PG, 3-phosphoglycerate PGK, 3-phospho- smallcaps" >d-glycerate kinase or ATP, 3-phospho- smallcaps" >d-glycerate 1-phosphotransferase (EC 2.7.2.3) hPGK, human PGK |
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