title: Substrate-induced conformational changes in Plasmodium falciparum guanosine monophosphate synthetase.
abstract: GMP synthetase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the nucleotide xanthosine 5'-monophosphate (XMP) to form guanosine 5'-monophosphate (GMP). Functional coordination of domains in glutamine amidotransferases leads to upregulation of glutamine hydrolysis in the presence of acceptor substrates and is a common feature in this class of enzymes. We have shown earlier that binding of substrates to the acceptor domain of Plasmodium falciparum GMP synthetase (PfGMPS) leads to enhancement in both glutaminase activity and rate of glutaminase inactivation, by the irreversible inhibitors acivicin and diazo-oxonorleucine [Bhat JY et al. (2008) Biochem J409, 263-273], a process that must be driven by conformational alterations. In this paper, through the combined use of biochemical assays, optical spectroscopy and mass spectrometry, we demonstrate that PfGMPS undergoes conformational transitions upon binding of substrates to the acceptor domain. Limited proteolysis and hydrogen-deuterium exchange in conjunction with mass spectrometry unveil region-specific conformational changes in the ATP + XMP bound state of PfGMPS. Decreased accessibility of R294 and K428 residues to trypsin in the ATP pyrophosphatase domain and reduced deuterium incorporation in the 143-155 region, pertaining to the glutaminase domain, suggest that in PfGMPS ligand-induced conformational changes are not only local but also transmitted over a long range across the domains. Overall, these results provide a detailed understanding of the substrate-induced changes in PfGMPS that could be essential for the overall catalytic process.
authors.affiliation: Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India.
authors.forename: Javaid Y
authors.initials: JY
authors.lastname: Bhat
authors.forename: Roopa
authors.initials: R
authors.lastname: Venkatachala
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Balaram
mesh_terms: D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D041961:Deuterium Exchange Measurement; D006157:Guanosine Monophosphate / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D010963:Plasmodium falciparum / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D015800:Protozoan Proteins; D006157:Guanosine Monophosphate; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing)