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 crystallographic studies.Comparative experiments with ADP
and MgADP have also been carried out. In contrast to the less specific
and 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 stabilize 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 metal complexes, however, behave differently. MgADP, but not MgATP, further increases theconformational stability with respect to its Mg
2+-free form, which indicates their different modes of bindingto the enzyme. Crystal structures of the binary complexes of the enzyme with MgATP
and with ATP (2.1
and 1.9 Å resolution, respectively) have shown that the orientation
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 metal 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 [Kov&
aacute;ri, Z., Flachner, B., N&
aacute;ray-Szabó, G.,
and Vas, M. (2002)
Biochemistry41, 8796-8806]. Mg
2+ is lig
anded to both
-
and -phosphates of ATP, while
-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 metal complexes of AMP-PNP with the N-terminus of helix 13
and with Asn336of
-str
and J are absent in the case of MgATP. Fluctuation of MgATP phosphates between two alternativesites has been proposed to facilitate the correct positioning of the mobile side chain of Lys215,
and thecatalytically competent active site is thereby completed.