Transition state analogue structures of human phosphoglycerate kinase establish the importance of charge balance in catalysis.

Transition state analogue (TSA) complexes formed by phosphoglycerate kinase (PGK) have been used to test the hypothesis that balancing of charge within the transition state dominates enzyme-catalyzed phosphoryl transfer. High-resolution structures of trifluoromagnesate (MgF(3)(-)) and tetrafluoroaluminate (AlF(4)(-)) complexes of PGK have been determined using X-ray crystallography and (19)F-based NMR methods, revealing the nature of the catalytically relevant state of this archetypal metabolic kinase. Importantly, the side chain of K219, which coordinates the alpha-phosphate group in previous ground state structures, is sequestered into coordinating the metal fluoride, thereby creating a charge environment complementary to the transferring phosphoryl group. In line with the dominance of charge balance in transition state organization, the substitution K219A induces a corresponding reduction in charge in the bound aluminum fluoride species, which changes to a trifluoroaluminate (AlF(3)(0)) complex. The AlF(3)(0) moiety retains the octahedral geometry observed within AlF(4)(-) TSA complexes, which endorses the proposal that some of the widely reported trigonal AlF(3)(0) complexes of phosphoryl transfer enzymes may have been misassigned and in reality contain MgF(3)(-).

[1]  C. Mathé,et al.  L-nucleoside enantiomers as antivirals drugs: a mini-review. , 2006, Antiviral research.

[2]  Fei Long,et al.  ARP/wARP and molecular replacement: the next generation , 2007, Acta crystallographica. Section D, Biological crystallography.

[3]  A. Leslie,et al.  Ground State Structure of F1-ATPase from Bovine Heart Mitochondria at 1.9 Å Resolution* , 2007, Journal of Biological Chemistry.

[4]  M. Williamson,et al.  Synthesis and binding of stable bisubstrate ligands for phosphoglycerate kinase. , 1998, Bioorganic & medicinal chemistry letters.

[5]  M. J. Parker,et al.  The development of tertiary interactions during the folding of a large protein. , 1996, Folding & design.

[6]  Beáta Flachner,et al.  Communication between the nucleotide site and the main molecular hinge of 3-phosphoglycerate kinase. , 2008, Biochemistry.

[7]  Aaron R. Dinner,et al.  Uracil-DNA glycosylase acts by substrate autocatalysis , 2001, Nature.

[8]  Winnie Yang,et al.  Phosphoglycerate kinase 1‐overexpressing lung cancer cells reduce cyclooxygenase 2 expression and promote anti‐tumor immunity in vivo , 2008, International journal of cancer.

[9]  Bradley E. Bernstein,et al.  Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation , 1997, Nature.

[10]  Katrin Rittinger,et al.  Structure at 1.65 Å of RhoA and its GTPase-activating protein in complex with a transition-state analogue , 1997, Nature.

[11]  Arieh Warshel,et al.  On the interpretation of the observed linear free energy relationship in phosphate hydrolysis: a thorough computational study of phosphate diester hydrolysis in solution. , 2008, Biochemistry.

[12]  O. Popanda,et al.  Modulation of DNA polymerases α, δ and ε by lactate dehydrogenase and 3-phosphoglycerate kinase , 1998 .

[13]  F. Westheimer.  Why nature chose phosphates. , 1987, Science.

[14]  R. Huber,et al.  Closed structure of phosphoglycerate kinase from Thermotoga maritima reveals the catalytic mechanism and determinants of thermal stability. , 1997, Structure.

[15]  A. Wittinghofer.  Signaling mechanistics: Aluminum fluoride for molecule of the year , 1997, Current Biology.

[16]  R. Martin.  Ternary complexes of Al3+ and F− with a third ligand , 1996 .

[17]  A. Fersht,et al.  Contribution of long-range electrostatic interactions to the stabilization of the catalytic transition state of the serine protease subtilisin BPN'. , 1993, Biochemistry.

[18]  T. Harris,et al.  High‐Precision Measurement of Hydrogen Bond Lengths in Proteins by Nuclear Magnetic Resonance Methods , 1999, Proteins.

[19]  W. Kabsch,et al.  The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. , 1997, Science.

[20]  I. Schlichting,et al.  pH influences fluoride coordination number of the AlFx phosphoryl transfer transition state analog , 1999, Nature Structural Biology.

[21]  J. Stivers,et al.  Powering DNA repair through substrate electrostatic interactions. , 2003, Biochemistry.

[22]  G. Lienhard,et al.  P 1 ,P 5 -Di(adenosine-5')pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase. , 1973, The Journal of biological chemistry.

[23]  N. J. Baxter,et al.  Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF 3 - rather than by phosphoranes , 2010, Proceedings of the National Academy of Sciences.

[24]  F. Hollfelder,et al.  A Trojan horse transition state analogue generated by MgF3− formation in an enzyme active site , 2006, Proceedings of the National Academy of Sciences.

[25]  Beáta Flachner,et al.  Role of phosphate chain mobility of MgATP in completing the 3-phosphoglycerate kinase catalytic site: binding, kinetic, and crystallographic studies with ATP and MgATP. , 2004, Biochemistry.

[26]  D. Hilvert,et al.  Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donor. , 2003, Journal of the American Chemical Society.

[27]  J. Szabó,et al.  Direct kinetic evidence that lysine 215 is involved in the phospho-transfer step of human 3-phosphoglycerate kinase. , 2009, Biochemistry.

[28]  Wei Yang,et al.  UvrD Helicase Unwinds DNA One Base Pair at a Time by a Two-Part Power Stroke , 2006, Cell.

[29]  S. Smerdon,et al.  MgF(3)(-) as a transition state analog of phosphoryl transfer. , 2002, Chemistry & biology.

[30]  A. Mathiowetz,et al.  Probing the role of arginines and histidines in the catalytic function and activation of yeast 3-phosphoglycerate kinase by site-directed mutagenesis. , 1990, The Journal of biological chemistry.

[31]  J. Tainer,et al.  Structure and mechanism of the RuvB Holliday junction branch migration motor. , 2001, Journal of molecular biology.

[32]  V. Schramm.  Enzymatic Transition State Theory and Transition State Analogue Design* , 2007, Journal of Biological Chemistry.

[33]  A. Warshel,et al.  Electrostatic basis for enzyme catalysis. , 2006, Chemical reviews.

[34]  P. Frey,et al.  Understanding enzymic catalysis: the importance of short, strong hydrogen bonds. , 1997, Chemistry & biology.

[35]  R. Raines,et al.  Pentavalent Organo-Vanadates as Transition State Analogues for Phosphoryl Transfer Reactions. , 2000, Journal of the American Chemical Society.

[36]  D. Flot,et al.  Expression, crystallization and X-ray data collection from microcrystals of the extracellular domain of the human inhibitory receptor expressed on myeloid cells IREM-1. , 2007, Acta crystallographica. Section F, Structural biology and crystallization communications.

[37]  R. Wolfenden,et al.  The rate of hydrolysis of phosphomonoester dianions and the exceptional catalytic proficiencies of protein and inositol phosphatases , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Elizabeth A. Gullen,et al.  Novel Role of 3-Phosphoglycerate Kinase, a Glycolytic Enzyme, in the Activation of L-Nucleoside Analogs, a New Class of Anticancer and Antiviral Agents* , 2003, Journal of Biological Chemistry.

[39]  R. Scopes.  The Steady‐State Kinetics of Yeast Phosphoglycerate Kinase , 1978 .

[40]  M. Vas,et al.  Insight into the mechanism of domain movements and their role in enzyme function: example of 3-phosphoglycerate kinase. , 2010, Current protein & peptide science.

[41]  A. Brunger.  Version 1.2 of the Crystallography and NMR system , 2007, Nature Protocols.

[42]  Richard J Morris,et al.  Towards complete validated models in the next generation of ARP/wARP. , 2004, Acta crystallographica. Section D, Biological crystallography.

[43]  J. Reinstein,et al.  Crystallization and preliminary X‐ray analysis of UMP/CMP‐kinase from Dictyostelium discoideum with the specific bisubstrate inhibitor P1‐(adenosine 5′)‐P5‐(uridine 5′)‐pentaphosphate (UP5A) , 1995, FEBS letters.

[44]  L. Pearl,et al.  Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery , 2004, The EMBO journal.

[45]  Beáta Flachner,et al.  Substrate‐induced double sided H‐bond network as a means of domain closure in 3‐phosphoglycerate kinase , 2006, FEBS letters.

[46]  M Ghosh,et al.  A 1.8 A resolution structure of pig muscle 3-phosphoglycerate kinase with bound MgADP and 3-phosphoglycerate in open conformation: new insight into the role of the nucleotide in domain closure. , 2001, Journal of molecular biology.

[47]  E. Scherzinger,et al.  Crystal structure of the hexameric replicative helicase RepA of plasmid RSF1010. , 2001, Journal of molecular biology.

[48]  Mohammad Reza Ahmadian,et al.  Confirmation of the arginine-finger hypothesis for the GAP-stimulated GTP-hydrolysis reaction of Ras , 1997, Nature Structural Biology.

[49]  E. Jaffe,et al.  Reactivity and metal-dependent stereospecificity of the phosphorothioate analogs of ADP and ATP and reactivity of Cr(III)ATP in the 3-phosphoglycerate kinase reaction. Structure of the metal nucleotide substrates. , 1982, The Journal of biological chemistry.

[50]  P. Hogg,et al.  Phosphoglycerate kinase acts in tumour angiogenesis as a disulphide reductase , 2000, Nature.

[51]  F. Hollfelder,et al.  Anionic charge is prioritized over geometry in aluminum and magnesium fluoride transition state analogs of phosphoryl transfer enzymes. , 2008, Journal of the American Chemical Society.

[52]  S. Shetty,et al.  Regulation of urokinase receptor expression by phosphoglycerate kinase is independent of its catalytic activity. , 2005, American journal of physiology. Lung cellular and molecular physiology.

[53]  W. Lam,et al.  Effect of hypoxia on the expression of phosphoglycerate kinase and antitumor activity of troxacitabine and gemcitabine in non-small cell lung carcinoma , 2009, Molecular Cancer Therapeutics.

[54]  F. Opperdoes.  Compartmentation of carbohydrate metabolism in trypanosomes. , 1987, Annual review of microbiology.

[55]  W. Cleland,et al.  Enzymatic mechanisms of phosphate and sulfate transfer. , 2006, Chemical reviews.

[56]  S. Arold,et al.  Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase , 2008, Nucleic acids research.

[57]  N. J. Baxter,et al.  MgF(3)(-) and alpha-galactose 1-phosphate in the active site of beta-phosphoglucomutase form a transition state analogue of phosphoryl transfer. , 2009, Journal of the American Chemical Society.

[58]  R. G. Kemp,et al.  Phosphofructo-1-kinase: role of charge neutralization in the active site. , 1995, Biochemical and biophysical research communications.

[59]  Beáta Flachner,et al.  Substrate-assisted movement of the catalytic Lys 215 during domain closure: site-directed mutagenesis studies of human 3-phosphoglycerate kinase. , 2005, Biochemistry.

[60]  Steven Hayward,et al.  Improvements in the analysis of domain motions in proteins from conformational change: DynDom version 1.50. , 2002, Journal of molecular graphics & modelling.