Kinetic characterization of adenylosuccinate synthetase from the thermophilic archaea Methanocaldococcus jannaschii.
Adenylosuccinate synthetase (AdSS) catalyzes the Mg2+ dependent condensation of a molecule of IMP with aspartate to form adenylosuccinate, in a reaction driven by the hydrolysis of GTP to GDP. AdSS from the thermophilic archaea, Methanocaldococcus jannaschii (MjAdSS) is 345 amino acids long against an average length of 430-457 amino acids for most mesophilic AdSS. This short AdSS has two large deletions that map to the middle and C-terminus of the protein. This article discusses the detailed kinetic characterization of MjAdSS. Initial velocity and product inhibition studies, carried out at 70 degrees C, suggest a rapid equilibrium random AB steady-state ordered C kinetic mechanism for the MjAdSS catalyzed reaction. AdSS are known to exhibit monomer-dimer equilibrium with the dimer being implicated in catalysis. In contrast, our studies show that MjAdSS is an equilibrium mixture of dimers and tetramers with the tetramer being the catalytically active form. The tetramer dissociates into dimers with a minor increase in ionic strength of the buffer, while the dimer is extremely stable and does not dissociate even at 1.2 M NaCl. Phosphate, a product of the reaction, was found to be a potent inhibitor of MjAdSS showing biphasic inhibition of enzyme activity. The inhibition was competitive with IMP and noncompetitive with GTP. MjAdSS, like the mouse acidic isozyme, exhibits substrate inhibition, with IMP inhibiting enzyme activity at subsaturating GTP concentrations. Regulation of enzyme activity by the glycolytic intermediate, fructose 1,6 bisphosphate, was also observed with the inhibition being competitive with IMP and noncompetitive against GTP.
Mehrotra
Sonali
S
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur Post, Bangalore 560064, India.
Balaram
Hemalatha
H
D000265:Adenylosuccinate Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D007700:Kinetics; D017017:Methanococcus / Q000201:enzymology*; D008954:Models, Biological; D020836:Protein Structure, Quaternary
D000265:Adenylosuccinate Synthase