Kinetic mechanism of glutathione synthetase from Arabidopsis thaliana.
Glutathione synthetase (GS) catalyzes the ATP-dependent formation of the ubiquitous peptide glutathione from gamma-glutamylcysteine and glycine. The bacterial and eukaryotic GS form two distinct families lacking amino acid sequence homology. Moreover, the detailed kinetic mechanism of the bacterial and the eukaryotic GS remains unclear. Here we have overexpressed Arabidopsis thaliana GS (AtGS) in an Escherichia coli expression system and purified the recombinant enzyme for biochemical characterization. AtGS is functional as a homodimeric protein with steady-state kinetic properties similar to those of other eukaryotic GS. The kinetic mechanism of AtGS was investigated using initial velocity methods and product inhibition studies. The best fit of the observed data was to the equation for a random Ter-reactant mechanism in which dependencies between the binding of some substrate pairs were preferred. The binding of either ATP or gamma-glutamylcysteine increased the binding affinity of AtGS for the other substrate by 10-fold. Likewise, the binding of ATP or glycine increased binding affinity for the other ligand by 3.5-fold. In contrast, binding of either glycine or gamma-glutamylcysteine causes a 6.7-fold decrease in binding affinity for the second molecule. Product inhibition studies suggest that ADP is the last product released from the enzyme. Overall, these observations are consistent with a random Ter-reactant mechanism for the eukaryotic GS in which the binding order of certain substrates is kinetically preferred for catalysis.
Jez
Joseph M
JM
Donald Danforth Plant Science Center, St. Louis, Missouri 63132, USA. jjez@danforthcenter.org
Cahoon
Rebecca E
RE
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D017360:Arabidopsis / Q000201:enzymology* / Q000378:metabolism; D001667:Binding, Competitive; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004151:Dipeptides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D005978:Glutathione; D005981:Glutathione Synthase / Q000737:chemistry* / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding
D018076:DNA, Complementary; D004151:Dipeptides; D010455:Peptides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005981:Glutathione Synthase; D005978:Glutathione; C017341:gamma-glutamylcysteine; D005998:Glycine