title: Kinetic mechanism of the dimeric ATP sulfurylase from plants.
abstract: In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5'-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PPi (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PPi in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively.
authors.affiliation: Department of Biology, Washington University in St. Louis, One Brookings Drive, St. Louis, MO 63130, U.S.A.
authors.forename: Geoffrey E
authors.initials: GE
authors.lastname: Ravilious
authors.forename: Jonathan
authors.initials: J
authors.lastname: Herrmann
authors.forename: Soon
authors.initials: S
authors.lastname: Goo Lee
authors.forename: Corey S
authors.initials: CS
authors.lastname: Westfall
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Jez
mesh_terms: D000250:Adenosine Phosphosulfate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D055162:Biocatalysis; D002704:Chlorates / Q000737:chemistry; D007700:Kinetics; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013025:Glycine max / Q000201:enzymology*; D013430:Sulfate Adenylyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013431:Sulfates / Q000737:chemistry
chemical_list: D002704:Chlorates; D010940:Plant Proteins; D013431:Sulfates; D000250:Adenosine Phosphosulfate; D000255:Adenosine Triphosphate; D013430:Sulfate Adenylyltransferase