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PDBsum entry 1vjc
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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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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Authors
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B.Flachner,
Z.Kovári,
A.Varga,
Z.Gugolya,
F.Vonderviszt,
G.Náray-Szabó,
M.Vas.
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Ref.
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Biochemistry, 2004,
43,
3436-3449.
[DOI no: ]
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PubMed id
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Abstract
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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.
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