Interaction of human 3-phosphoglycerate kinase with L-ADP, the mirror image of D-ADP.
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.
Varga
Andrea
A
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Karolina Str. 29, P.O. Box 7, H-1518 Budapest, Hungary.
Szabó
Judit
J
Flachner
Beáta
B
Roy
Béatrice
B
Konarev
Peter
P
Svergun
Dmitri
D
Závodszky
Péter
P
Périgaud
Christian
C
Barman
Tom
T
Lionne
Corinne
C
Vas
Mária
M
D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010735:Phosphoglycerate Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D053838:Scattering, Small Angle; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014961:X-Ray Diffraction
D004163:Diphosphoglyceric Acids; D004791:Enzyme Inhibitors; D013438:Sulfhydryl Compounds; C015891:glycerate 1,3-biphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase; D008274:Magnesium