Kinetic and biochemical characterization of Plasmodium falciparum GMP synthetase.
Plasmodium falciparum, the causative agent of the fatal form of malaria, synthesizes GMP primarily from IMP and, hence, needs active GMPS (GMP synthetase) for its survival. GMPS, a G-type amidotransferase, catalyses the amination of XMP to GMP with the reaction occurring in two domains, the GAT (glutamine amidotransferase) and ATPPase (ATP pyrophosphatase). The GAT domain hydrolyses glutamine to glutamate and ammonia, while the ATPPase domain catalyses the formation of the intermediate AMP-XMP from ATP and XMP. Co-ordination of activity across the two domains, achieved through channelling of ammonia from GAT to the effector domain, is the hallmark of amidotransferases. Our studies aimed at understanding the kinetic mechanism of PfGMPS (Plasmodium falciparum GMPS) indicated steady-state ordered binding of ATP followed by XMP to the ATPPase domain with glutamine binding in a random manner to the GAT domain. We attribute the irreversible, Ping Pong step seen in initial velocity kinetics to the release of glutamate before the attack of the adenyl-XMP intermediate by ammonia. Specific aspects of the overall kinetic mechanism of PfGMPS are different from that reported for the human and Escherichia coli enzymes. Unlike human GMPS, absence of tight co-ordination of activity across the two domains was evident in the parasite enzyme. Variations seen in the inhibition by nucleosides and nucleotide analogues between human GMPS and PfGMPS highlighted differences in ligand specificity that could serve as a basis for the design of specific inhibitors. The present study represents the first report on recombinant His-tagged GMPS from parasitic protozoa.
Bhat
Javaid Yousuf
JY
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore-560064, India.
Shastri
Brahmanaspati Ganapathi
BG
Balaram
Hemalatha
H
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D005816:Genetic Complementation Test; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D010963:Plasmodium falciparum / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry
D008024:Ligands; D005973:Glutamine; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing)