Publication Date:
2013-07-10
Description:
In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyzes the thermodynamically unfavorable formation of a mixed phosphosulfate anhydride in adenosine 5’-phosphosulfate (APS) 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. Because of these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase (GmATPS1). 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. Isothermal titration calorimetry showed tight binding of APS to GmATPS1. In contrast, binding of PP i to GmATPS1 was not detected, although titration of the E•APS complex with PP i 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.
Print ISSN:
0144-8463
Electronic ISSN:
1573-4935
Topics:
Biology
,
Chemistry and Pharmacology
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