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 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. (c) 2007 Elsevier Inc. All rights reserved.