The complexes of pig muscle 3-phosphoglycerate kinase with the substrate MgATP and withthe nonsubstrate Mg
2+-free ATP have been characterized by binding, kinetic, and crys
tallographic studies.Comparative experiments with ADP and MgADP have also been carried out. In contrast to the less specificand largely ionic binding of Mg
2+-free ATP and ADP, specific occupation of the adenosine binding pocketby MgATP and MgADP has been revealed by displacement experiments with adenosine and anions, aswell as supported by isothermal calorimetric titrations. The Mg
2+-free nucleotides similarly s
tabilize theoverall protein structure and restrict the conformational flexibility around the reactive thiol groups ofhelix 13, as observed by differential scanning microcalorimetry and thiol reactivity studies, respectively.The me
tal complexes, however, behave differently. MgADP, but not MgATP, further increases theconformational s
tability with respect to its Mg
2+-free form, which indicates their different modes of bindingto the enzyme. Crys
tal structures of the binary complexes of the enzyme with MgATP and with ATP (2.1and 1.9 Å resolution, respectively) have shown that the orien
tation and interaction of phosphates of MgATPlargely differ not only from those of ATP but also from the previously determined ones of either MgADP[Davies, G. J., Gamblin, S. J., Littlechild, J. A., Dauter, Z., Wilson, K. S., and Watson, H. C. (1994)
ActaCrystallogr. D50, 202-209] or the me
tal complexes of AMP-PNP [May, A., Vas, M., Harlos, K., andBlake, C. C. F. (1996)
Proteins 24, 292-303; Auerbach, G., Huber, R., Grattinger, M., Zaiss, K., Schurig,H., Jaenicke, R., and Jacob, U. (1997)
Structure 5, 1475-1483] and are more similar to the interactionsformed with MgAMP-PCP [Kovári, Z.,
Flachner, B., Náray-Szabó, G., and Vas, M. (2002)
Biochemistry41, 8796-8806]. Mg
2+ is liganded to both
ta2.gif" BORDER=0 ALIGN="middle">- and
-phosphates of ATP, while
ta2.gif" BORDER=0 ALIGN="middle">-phosphate is linked tothe conserved Asp218, i.e., to the N-terminus of helix 8, through a water molecule; the known interactionsof either MgADP or the me
tal complexes of AMP-PNP with the N-terminus of helix 13 and with Asn336of
ta2.gif" BORDER=0 ALIGN="middle">-strand J are absent in the case of MgATP. Fluctuation of MgATP phosphates between two alternativesites has been proposed to facili
tate the correct positioning of the mobile side chain of Lys215, and theca
talytically competent active site is thereby completed.