title: Ammonia channeling in Plasmodium falciparum GMP synthetase: investigation by NMR spectroscopy and biochemical assays.
abstract: GMP synthetase, a class I amidotransferase, catalyzes the last step of the purine biosynthetic pathway, where ammonia from glutamine is incorporated into xanthosine 5'-monophospate to yield guanosine 5'-monnophosphate as the main product. Combined biochemical, structural, and computational studies of glutamine amidotransferases have revealed the existence of physically separate active sites connected by molecular tunnels that efficiently transfer ammonia from the glutaminase site to the synthetase site. Here, we have investigated aspects of ammonia channeling in P. falciparum GMP synthetase using biochemical assays in conjunction with 15N-edited proton NMR spectroscopy. Our results suggest that (1) ammonia released from glutamine is not equilibrated with the external medium, (2) saturating concentrations of glutamine do not obliterate the incorporation of external ammonia into GMP, and (3) ammonia in the external medium can access the thioester intermediate when the ATPPase domain is bound to substrates. Further, mutation of Cys-102 to alanine confirmed its identity as the catalytic residue in the glutaminase domain, and ammonia-dependent assays on the mutant indicated glutamine to be a partial uncompetitive inhibitor of the enzyme.
authors.affiliation: Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India.
authors.forename: Javaid Yousuf
authors.initials: JY
authors.lastname: Bhat
authors.forename: Roopa
authors.initials: R
authors.lastname: Venkatachala
authors.forename: Kavita
authors.initials: K
authors.lastname: Singh
authors.forename: Kallol
authors.initials: K
authors.lastname: Gupta
authors.forename: Siddhartha P
authors.initials: SP
authors.lastname: Sarma
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Balaram
mesh_terms: D000641:Ammonia / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007555:Isoxazoles / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D010963:Plasmodium falciparum / Q000201:enzymology
chemical_list: D007555:Isoxazoles; D009587:Nitrogen Isotopes; D005973:Glutamine; D000641:Ammonia; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing); C006303:acivicin