title: Studies on active site mutants of P. falciparum adenylosuccinate synthetase: insights into enzyme catalysis and activation.
abstract: Adenylosuccinate synthetase catalyzes a reversible reaction utilizing IMP, GTP and aspartate in the presence of Mg²+ to form adenylosuccinate, GDP and inorganic phosphate. Comparison of similarly liganded complexes of Plasmodium falciparum, mouse and Escherichia coli AdSS reveals H-bonding interactions involving nonconserved catalytic loop residues (Asn429, Lys62 and Thr307) that are unique to the parasite enzyme. Site-directed mutagenesis has been used to examine the role of these interactions in catalysis and structural organization of P. falciparum adenylosuccinate synthetase (PfAdSS). Mutation of Asn429 to Val, Lys62 to Leu and Thr307 to Val resulted in an increase in K(m) values for IMP, GTP and aspartate, respectively along with a 5 fold drop in the k(cat) value for N429V mutant suggesting the role of these residues in ligand binding and/or catalysis. We have earlier shown that the glycolytic intermediate, fructose 1,6 bisphosphate, which is an inhibitor of mammalian AdSS is an activator of the parasite enzyme. Enzyme kinetics along with molecular docking suggests a mechanism for activation wherein F16BP seems to be binding to the Asp loop and inducing a conformation that facilitates aspartate binding to the enzyme active site. Like in other AdSS, a conserved arginine residue (Arg155) is involved in dimer crosstalk and interacts with IMP in the active site of the symmetry related subunit of PfAdSS. We also report on the biochemical characterization of the arginine mutants (R155L, R155K and R155A) which suggests that unlike in E. coli AdSS, Arg155 in PfAdSS influences both ligand binding and catalysis.
authors.affiliation: Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India.
authors.forename: Sonali
authors.initials: S
authors.lastname: Mehrotra
authors.forename: B N
authors.initials: BN
authors.lastname: Mylarappa
authors.forename: Prathima
authors.initials: P
authors.lastname: Iyengar
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Balaram
mesh_terms: D000265:Adenylosuccinate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology*; D011487:Protein Conformation
chemical_list: D050505:Mutant Proteins; D001120:Arginine; D000265:Adenylosuccinate Synthase