Substrate-assisted movement of the catalytic Lys 215 during domain closure: site-directed mutagenesis studies of human 3-phosphoglycerate kinase.

3-Phosphoglycerate kinase (PGK) is a two-domain hinge-bending enzyme. It is still unclear how the geometry of the active site is formed during domain closure and how the catalytic residues are brought into the optimal position for the reaction. Comparison of the three-dimensional structures in various open and closed conformations suggests a large (10 A) movement of Lys 215 during domain closure. This change would be required for direct participation of this side chain in both the catalyzed phospho transfer and the special anion-caused activation. To test the multiple roles of Lys 215, two mutants (K215A and K215R) were constructed from human PGK and characterized in enzyme kinetic and substrate binding studies. For comparison, mutants (R38A and R38K) of the known essential residue, Arg 38, were also produced. Drastic decreases (1500- and 500-fold, respectively), as in the case of R38A, were observed in the kcat values of mutants K215A and K215R, approving the essential catalytic role of Lys 215. In contrast, the R38K mutation caused an only 1.5-fold decrease in activity. This emphasizes the importance of a very precise positioning of Lys 215 in the active site, in addition to its positive charge. The side chain of Lys 215 is also responsible for the substrate and anion-dependent activation, since these properties are abolished upon mutation. Among the kinetic constants mainly the Km values of MgATP and 1,3-BPG are increased (approximately 20- and approximately 8-fold, respectively) in the case of the neutral K215A mutant, evidence of the interaction of Lys 215 with the transferring phospho group in the functioning complex. Weakening of MgATP binding (a moderate increase in Kd), but not of MgADP binding, upon mutation indicates an initial weak interaction of Lys 215 with the gamma-phosphate already in the nonfunctioning open conformation. Thus, during domain closure, Lys 215 possibly moves together with the transferring phosphate; meanwhile, this group is being positioned properly for catalysis.

[1]  Richard D. Taylor,et al.  Improved protein–ligand docking using GOLD , 2003, Proteins.

[2]  L. Johnson,et al.  Activator anion binding site in pyridoxal phosphorylase b: The binding of phosphite, phosphate, and fluorophosphate in the crystal , 1996, Protein science : a publication of the Protein Society.

[3]  D. Watts,et al.  Anion activation of monkey muscle creatine kinase. , 1984, The International journal of biochemistry.

[4]  K. Harlos,et al.  2.0 Å resolution structure of a ternary complex of pig muscle phosphoglycerate kinase containing 3‐phospho‐D‐glycerate and the nucleotide Mn adenylylimidodiphosphate , 1996, Proteins.

[5]  K Harlos,et al.  Crystal structure of the binary complex of pig muscle phosphoglycerate kinase and its substrate 3‐phospho‐D‐glycerate , 1992, Proteins.

[6]  Michel Véron,et al.  Broad specificity of human phosphoglycerate kinase for antiviral nucleoside analogs. , 2004, Biochemical pharmacology.

[7]  A. Riggs,et al.  Evolutionary conservation of the substrate‐binding cleft of phosphoglycerate kinases , 1986, FEBS Letters.

[8]  Y. S. Brownstone,et al.  A study of phosphoglycerate kinase in human erythrocytes. II. Kinetic properties. , 1976, Biochimica et biophysica acta.

[9]  M. Larsson-Raźnikiewicz.  Kinetic studies on the reaction catalyzed by phosphoglycerate kinase. II. The kinetic relationships between 3-phosphoglycerate, MgATP2-and activating metal ion. , 1964, Biochimica et biophysica acta.

[10]  L. Becker,et al.  Functional coupling between glycolysis and sarcoplasmic reticulum Ca2+ transport. , 1995, Circulation research.

[11]  C. Craescu,et al.  Critical Role of Human Bisphosphoglycerate Mutase Cys22 in the Phosphatase Activator-binding Site* , 1997, The Journal of Biological Chemistry.

[12]  Crystallographic and thiol-reactivity studies on the complex of pig muscle phosphoglycerate kinase with ATP analogues: correlation between nucleotide binding mode and helix flexibility. , 2002, Biochemistry.

[13]  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.

[14]  K. Mizumoto,et al.  Involvement of a Cellular Glycolytic Enzyme, Phosphoglycerate Kinase, in Sendai Virus Transcription* , 1999, The Journal of Biological Chemistry.

[15]  R. Gupta,et al.  Measurement of the dissociation constant of MgATP at physiological nucleotide levels by a combination of 31P NMR and optical absorbance spectroscopy. , 1983, Biochemical and biophysical research communications.

[16]  M. Williamson,et al.  Highly potent bisphosphonate ligands for phosphoglycerate kinase. , 1998, Journal of medicinal chemistry.

[17]  A. Mathiowetz,et al.  Site-directed mutations of arginine 65 at the periphery of the active site cleft of yeast 3-phosphoglycerate kinase enhance the catalytic activity and eliminate anion-dependent activation. , 1991, Protein engineering.

[18]  T. McPhillips,et al.  Structure of the R65Q mutant of yeast 3-phosphoglycerate kinase complexed with Mg-AMP-PNP and 3-phospho-D-glycerate. , 1996, Biochemistry.

[19]  R. Stinson,et al.  Anion binding to yeast phosphoglycerate kinase. , 1978, European journal of biochemistry.

[20]  J. Vishwanatha,et al.  The role of primer recognition proteins in DNA replication: association with nuclear matrix in HeLa cells. , 1992, Journal of cell science.

[21]  E. Serpersu,et al.  A new metal-binding site for yeast phosphoglycerate kinase as determined by the use of a metal-ATP analog. , 1997, Biophysical journal.

[22]  Wing Lam,et al.  Phosphorylation of Pyrimidine Deoxynucleoside Analog Diphosphates , 2002, The Journal of Biological Chemistry.

[23]  J. D. Gregory,et al.  Arrangement of substrates at the active site of yeast phosphoglycerate kinase. Effect of sulfate ion. , 1993, The Journal of biological chemistry.

[24]  H. Watson,et al.  Characterisation of yeast phosphoglycerate kinase modified by mutagenesis at residue 21. , 1992, European journal of biochemistry.

[25]  G. Kürzinger,et al.  The reconstituted ADP/ATP carrier from mitochondria is both inhibited and activated by anions. , 1984, Biochimica et biophysica acta.

[26]  D. Harris,et al.  Studies of the kinetics of the isolated mitochondrial ATPase using dinitrophenol as a probe. , 1981, Biochimica et biophysica acta.

[27]  P. Tompa,et al.  The phosphate group of 3-phosphoglycerate accounts for conformational changes occurring on binding to 3-phosphoglycerate kinase. Enzyme inhibition and thiol reactivity studies. , 1986, European journal of biochemistry.

[28]  P. Krishnan,et al.  Phosphorylation of Pyrimidine l-Deoxynucleoside Analog Diphosphates , 2002, The Journal of Biological Chemistry.

[29]  P. Hogg,et al.  Disulfide bonds as switches for protein function. , 2003, Trends in biochemical sciences.

[30]  M. Larsson-Raźnikiewicz,et al.  Activation and inhibition of phosphoglycerate kinase by sulphate ion. , 1981, Biochimica et biophysica acta.

[31]  D. Lüthi,et al.  Apparent Mg2+-adenosine 5-triphosphate dissociation constant measured with Mg2+ macroelectrodes under conditions pertinent to 31P NMR ionized magnesium determinations. , 1997, Analytical biochemistry.

[32]  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.

[33]  B. Bernstein,et al.  Crystal structures of substrates and products bound to the phosphoglycerate kinase active site reveal the catalytic mechanism. , 1998, Biochemistry.

[34]  O. Ptitsyn,et al.  Correlation between enzyme activity and hinge-bending domain displacement in 3-phosphoglycerate kinase. , 1989, European journal of biochemistry.

[35]  M. Vas,et al.  Effects of substrates on the heat stability and on the reactivities of thiol groups of 3-phosphoglycerate kinase. , 1983, European journal of biochemistry.

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

[37]  H. Wu,et al.  Suppression of angiogenesis and tumor growth by the inhibitor K1-5 generated by plasmin-mediated proteolysis. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[38]  T. Steitz,et al.  Substrate binding closes the cleft between the domains of yeast phosphoglycerate kinase. , 1979, The Journal of biological chemistry.

[39]  I. Mavelli,et al.  Effects of Fe(III) binding to the nucleotide‐independent site of F1‐ATPase: enzyme thermostability and response to activating anions , 2001, FEBS letters.

[40]  R. J. Williams,et al.  An NMR study of anion binding to yeast phosphoglycerate kinase. , 1990, European journal of biochemistry.

[41]  H. Watson,et al.  Isoenzymes of phosphoglycerate kinase: evolutionary conservation of the structure of this glycolytic enzyme. , 1990, Biochemical Society transactions.

[42]  J. Riordan,et al.  Sulfate potentiation of the chloride activation of angiotensin converting enzyme. , 1987, Biochemistry.

[43]  H. Watson,et al.  Site-directed mutagenesis of yeast phosphoglycerate kinase. The 'basic-patch' residue arginine 168. , 1989, European journal of biochemistry.

[44]  P. Hogg,et al.  Secretion of phosphoglycerate kinase from tumour cells is controlled by oxygen-sensing hydroxylases. , 2004, Biochimica et biophysica acta.

[45]  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.

[46]  M. Vas,et al.  Anion activation of 3-phosphoglycerate kinase requires domain closure. , 1998, Biochemistry.

[47]  N. Calcaterra,et al.  Steady‐state kinetics of F1‐ATPase , 1985, FEBS letters.

[48]  P. Hogg,et al.  Plasmin Reduction by Phosphoglycerate Kinase Is a Thiol-independent Process* , 2002, The Journal of Biological Chemistry.

[49]  R. Stinson,et al.  The effects of anions, substrates, metal ions and sulfhydryl reagents on the proteolytic susceptibility of yeast phosphoglycerate kinase. , 1981, Biochimica et biophysica acta.

[50]  A. Lavoinne,et al.  Kinetic studies of the reaction mechanism of rat liver phosphoglycerate kinase in the direction of ADP utilization. , 1983, Biochimie.

[51]  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.

[52]  B. Zerner,et al.  [8] Reassessment of Ellman's reagent , 1983 .

[53]  S. Velick,et al.  THE ACYL-ENZYME INTERMEDIATE AND THE KINETIC MECHANISM OF THE GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE REACTION. , 1965, The Journal of biological chemistry.

[54]  G J Davies,et al.  Structure of the ADP complex of the 3-phosphoglycerate kinase from Bacillus stearothermophilus at 1.65 A. , 1994, Acta crystallographica. Section D, Biological crystallography.

[55]  H. Watson,et al.  Site‐directed mutagenesis of histidine 62 in the ‘basic patch’ region of yeast phosphoglycerate kinase , 1989, FEBS letters.

[56]  W. Fairbrother,et al.  Characterization of the structure and properties of the His 62 → Ala and Arg 38 → Ala mutants of yeast phosphoglycerate kinase: An investigation of the catalytic and activatory sites by site‐directed mutagenesis and NMR , 1992, Protein science : a publication of the Protein Society.

[57]  M. Williamson,et al.  Orientation of 1,3-Bisphosphoglycerate Analogs Bound to Phosphoglycerate Kinase* , 2003, Journal of Biological Chemistry.

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

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

[60]  P. Evans,et al.  Sequence, structure and activity of phosphoglycerate kinase: a possible hinge-bending enzyme , 1979, Nature.

[61]  R. Scopes.  Binding of substrates and other anions to yeast phosphoglycerate kinase. , 1978, European journal of biochemistry.

[62]  P. Hogg,et al.  Generation of Angiostatin by Reduction and Proteolysis of Plasmin , 1997, The Journal of Biological Chemistry.

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

[64]  B. Glader,et al.  Energy metabolism in human erythrocytes: the role of phosphoglycerate kinase in cation transport. , 1975, Blood.

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

[66]  J. Stuehr,et al.  Interactions of divalent metal ions with inorganic and nucleoside phosphates. I. Thermodynamics. , 1972, Journal of the American Chemical Society.