The complexes of pi
g muscle 3-phospho
glycerate kinase with the substrate M
gATP
and withthe nonsubstrate M
g2+-free ATP have been characterized by bindin
g, kinetic,
and crystallo
graphic studies.Comparative experiments with ADP
and M
gADP have also been carried out. In contrast to the less specific
and lar
gely ionic bindin
g of M
g2+-free ATP
and ADP, specific occupation of the adenosine bindin
g pocketby M
gATP
and M
gADP has been revealed by displacement experiments with adenosine
and anions, aswell as supported by isothermal calorimetric titrations. The M
g2+-free nucleotides similarly stabilize theoverall protein structure
and restrict the conformational flexibility around the reactive thiol
groups ofhelix 13, as observed by differential scannin
g microcalorimetry
and thiol reactivity studies, respectively.The metal complexes, however, behave differently. M
gADP, but not M
gATP, further increases theconformational stability with respect to its M
g2+-free form, which indicates their different modes of bindin
gto the enzyme. Crystal structures of the binary complexes of the enzyme with M
gATP
and with ATP (2.1
and 1.9 &Arin
g; resolution, respectively) have shown that the orientation
and interaction of phosphates of M
gATPlar
gely differ not only from those of ATP but also from the previously determined ones of either M
gADP[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., Grattin
ger, M., Zaiss, K., Schuri
g,H., Jaenicke, R.,
and Jacob, U. (1997)
Structure 5, 1475-1483]
and are more similar to the interactionsformed with M
gAMP-PCP [Kov&
aacute;ri, Z., Flachner, B., N&
aacute;ray-Szabó, G.,
and Vas, M. (2002)
Biochemistry41, 8796-8806]. M
g2+ is li
ganded to both
ges/
gifchars/beta2.
gif" BORDER=0 ALIGN="middle">-
and ges/
gifchars/
gamma.
gif" BORDER=0 >-phosphates of ATP, while
ges/
gifchars/beta2.
gif" BORDER=0 ALIGN="middle">-phosphate is linked tothe conserved Asp218, i.e., to the N-terminus of helix 8, throu
gh a water molecule; the known interactionsof either M
gADP or the metal complexes of AMP-PNP with the N-terminus of helix 13
and with Asn336of
ges/
gifchars/beta2.
gif" BORDER=0 ALIGN="middle">-str
and J are absent in the case of M
gATP. Fluctuation of M
gATP phosphates between two alternativesites has been proposed to facilitate the correct positionin
g of the mobile side chain of Lys215,
and thecatalytically competent active site is thereby completed.