ATP sulfurylase, from Escherichia coli K-12, catalyzes both the hydrolysis of GTP and the synthesis of activated sulfate (APS). This paper describes the energetic linkage of these reactions and the events that couple them. Steady-state and single-turnover experiments suggest that the binding of GTP inhibits APS production and that the hydrolysis of GTP is required to generate the enzyme form(s) that produces APS. It is this progression from the inhibitory, E GTP, to the productive, E GDP, complexes in the cycle of APS synthesis that energetically links these two reactions. This model stands in contrast to other GTPase/target systems in which the binding of GTP alone is sufficient to catalyze multiple turnovers of the target reaction. The stoichiometry of GTP hydrolysis to APS synthesis is 1:1, and equilibrium measurements show that −9.1 kcal/mol, produced by the hydrolysis of GTP, is used to thermodynamically drive production of APS and PPi. These findings establish the mechanism of energy transfer in this novel GTPase/target system, and substantially alter our understanding of the energetics of sulfate activation, an essential step in the metabolic assimilation of sulfur.
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