Role of Phosphate Chain Mobility of MgATP in Completing the 3-Phosphoglycerate Kinase Catalytic Site:  Binding, Kinetic, and Crystallographic Studies with ATP and MgATP

Abstract

The complexes of pig muscle 3-phosphoglycerate kinase with the substrate MgATP and with the 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 pocket by MgATP and MgADP has been revealed by displacement experiments with adenosine and anions, as well as supported by isothermal calorimetric titrations. The Mg(2+)-free nucleotides similarly stabilize the overall protein structure and restrict the conformational flexibility around the reactive thiol groups of helix 13, as observed by differential scanning microcalorimetry and thiol reactivity studies, respectively. The metal complexes, however, behave differently. MgADP, but not MgATP, further increases the conformational stability with respect to its Mg(2+)-free form, which indicates their different modes of binding to the enzyme. Crystal structures of the binary complexes of the enzyme with MgATP and with ATP (2.1 and 1.9 A resolution, respectively) have shown that the orientation and interaction of phosphates of MgATP largely 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) Acta Crystallogr. D50, 202-209] or the metal complexes of AMP-PNP [May, A., Vas, M., Harlos, K., and Blake, 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 interactions formed with MgAMP-PCP [Kovári, Z., Flachner, B., Náray-Szabó, G., and Vas, M. (2002) Biochemistry 41, 8796-8806]. Mg(2+) is liganded to both beta- and gamma-phosphates of ATP, while beta-phosphate is linked to the conserved Asp218, i.e., to the N-terminus of helix 8, through a water molecule; the known interactions of either MgADP or the metal complexes of AMP-PNP with the N-terminus of helix 13 and with Asn336 of beta-strand J are absent in the case of MgATP. Fluctuation of MgATP phosphates between two alternative sites has been proposed to facilitate the correct positioning of the mobile side chain of Lys215, and the catalytically competent active site is thereby completed.

A Spring-loaded Release Mechanism Regulates Domain Movement and Catalysis in Phosphoglycerate Kinase

“…The model obtained gives an excellent fit to the data (Table 1, coordinates available in supplemental material) and shows a conformation considerably more open than observed in crystal structures, with a 56°rota-tion required to form the catalytically active conformation (as opposed to a maximum of 33°from the most open crystal structure ( Fig. 2B) (37). The validity of the model produced is supported molecular dynamics studies (16) that detected a further opening of PGK, when compared with "open" crystal structures.…”

Structure-Based Mutagenesis of Sulfolobus Turreted Icosahedral Virus B204 Reveals Essential Residues in the Virion-Associated DNA-Packaging ATPase

“…1b). These differences in ␥-phosphate orientation could be attributed to the utilization of different ATP analogs (AMP-PNP for STIV B204 and AMP-PCP for STIV2 B204) in crystallization experiments (35) or to the absence of a divalent cation in crystal structures of STIV B204. Despite the lack of metal ion supplementation in crystallization experiments, a tetrahedrally coordinated zinc ion was observed in both apo and AMP-PNP-bound crystal structures in the same location as that observed for structures of STIV2 B204 (21).…”

Structural Insights into the Catalytic Mechanism of Phosphate Ester Hydrolysis by dUTPase

“…In fact, in the dUTPase:dUDP:Sr 2ϩ complex, the critical position of the ␣-phosphorus is 3.2 Å away from its competent site as determined in our present dUTPase:dUTP:Mg 2ϩ and Mg 2ϩ :␣,␤-imino-dUTP:dUTPase structures. The coordination of Mg 2ϩ to all the three phosphate groups is not very common in other nucleotide-protein structures where the metal ion is frequently seen as coordinating to only two phosphate groups (41)(42)(43)(44). In the dUTPase structures, the triple phosphate coordination of Mg 2ϩ contributes to an increasingly compact phosphate chain conformation presumably optimal for catalysis.…”