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

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• 作者：Beá ; ta Flachner ; Zoltá ; n Ková ; ri ; Andrea Varga ; Zoltá ; n Gugolya ; Ferenc Vonderviszt ; Gá ; bor Ná ; ray-Szabó ; and Má ; ria Vas
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：March 30, 2004
• 年：2004
• 卷：43
• 期：12
• 页码：3436 - 3449
• 全文大小：898K
• 年卷期：v.43,no.12(March 30, 2004)
• ISSN：1520-4995

文摘

The complexes of pig muscle 3-phosphoglycerate kinase with the substrate MgATP and withthe nonsubstrate Mg²⁺-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 specificand largely ionic binding of Mg²⁺-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 Mg²⁺-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 Mg²⁺-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 (2.1and 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 [Kovári, Z., Flachner, B., Náray-Szabó, G., and Vas, M. (2002) Biochemistry41, 8796-8806]. Mg²⁺ is liganded 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 -strand 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.