Binding of nucleotides to guanylate kinase, p21(ras), and nucleoside-diphosphate kinase studied by nano-electrospray mass spectrometry.
The binding of nucleotides to three different nucleotide-binding proteins and to a control protein was studied by means of nano-electrospray mass spectrometry applied to aqueous nondenaturing solutions. The method leads to unambiguous identification of enzyme complexes with substrates and products but does not allow the determination of dissociation constants or even stoichiometries relevant to the binding in solution. For guanylate kinase (EC 2.7.4. 8), the transfer of HPO(3) between nucleotides was observed whenever a ternary complex with adenylate or guanylate nucleotides was formed. Guanosine 5'-tetraphosphate was generated after prolonged incubation with GDP or GTP. Mg(2+) binding was considerably enhanced in functional high affinity complexes, such as observed between guanylate kinase and its bisubstrate inhibitor P(1)-(5'-guanosyl)-P(5)-(5'-adenosyl) pentaphosphate or with the tight nucleotide-binding protein p21(ras) and GDP. Nucleoside-diphosphate kinase (EC 2.7.4.6) itself was phosphorylated in accordance to its known ping-pong mechanism. All nucleotide-binding proteins were shown to bind sulfate (SO(4)(2-)) with presumably high affinity and slow exchange rate. The binding of phosphate (PO(4)(3-)) could be inferred indirectly from competition with SO(4)(2-).
Prinz
H
H
Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Str. 11, D-44227 Dortmund, Germany. heino.prinz@mpi-dortmund.mpg.de
Lavie
A
A
Scheidig
A J
AJ
Spangenberg
O
O
Konrad
M
M
D001665:Binding Sites; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D051528:Guanylate Kinases; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000378:metabolism*; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013431:Sulfates / Q000378:metabolism
D006150:Guanine Nucleotides; D010710:Phosphates; D011994:Recombinant Proteins; D013431:Sulfates; D006153:Guanosine Diphosphate; D006159:Guanosine Tetraphosphate; D006160:Guanosine Triphosphate; D009703:Nucleoside-Phosphate Kinase; D009701:Nucleoside-Diphosphate Kinase; D051528:Guanylate Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)