Effect of metal-ligand mutations on phosphoryl transfer reactions catalyzed by Escherichia coli glutamine synthetase.
Glutamine synthetase (GS) converts glutamate to glutamine in the presence of ATP and ammonia and requires two divalent metal ions, designated n1 and n2, for catalysis. The first intermediate, gamma-glutamyl phosphate, is formed during catalysis by the transfer of the gamma-phosphate of ATP to the gamma-carboxylate of glutamate. Efficient phosphoryl transfer between these two negatively charged moieties is thought to be mediated by the n2 metal. To explore the role of the n2 metal in catalysis, histidine 269, a ligand to the n2 metal, was changed to aspartate, asparagine, glutamate, and glutamine by site-directed mutagenesis. All of the mutants bind two manganese ions as determined by EPR titration. The mutations had little effect on the substrate Km's except in the case of H269E which exhibited a Km Glu = 92 mM, a 1000-fold increase compared to that for WT (Km Glu = 70 microM). The ability of these mutants to catalyze phosphoryl transfer to glutamate or to the inhibitor phosphinothricin was examined by rapid quench kinetic experiments. Phosphorylated phosphinothricin was detected by 31P NMR and shown to be produced by both mutants and WT. The rate of phosphoryl transfer to PPT for H269E is reduced 100-fold (0.030 s-1) compared to WT (8 s-1). The extra negative charge around the n2 metal ion contributed by glutamate 269 severely reduces the ability of the n2 metal to mediate efficient glutamate binding in the presence of negatively charged ATP and weakens the interactions between metal ion and the reactants in the transition state, thus resulting in a lower rate of phosphoryl transfer.
Abell
L M
LM
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
Schineller
J
J
Keck
P J
PJ
Villafranca
J J
JJ
D000595:Amino Acid Sequence; D000613:Aminobutyrates / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation
D000613:Aminobutyrates; D004269:DNA, Bacterial; D008024:Ligands; D008670:Metals; D005973:Glutamine; D018698:Glutamic Acid; D008345:Manganese; C003121:phosphinothricin; D005974:Glutamate-Ammonia Ligase