ATP sulfurylase, from Escherichia coli K-12, conformationally couples the rates and chemical potentials of the two reactions that it catalyzes, GTP hydrolysis and activated sulfate synthesis. The enzyme is rare among such coupling systems in that it links the potentials of small-molecule chemistries to one another, rather than to vectorial motion. The pre-steady-state stages of the catalytic cycle of ATP sulfurylase were studied using tools capable of distinguishing between enzyme-bound and solution-phase product for each of the four products of the enzyme. The study reveals that the two chemistries are linked at multiple points in the reaction coordinate. Linking begins with an isomerization prior to chemistry that initiates an ordered cleavage of the β,γ and α,β bonds of GTP and ATP, respectively; the rates of these three sequential events increase successively, causing them to appear simultaneous. Linking is again seen in the late stages of the catalytic cycle: product release is ordered with Pi departing prior to either GDP or PPi. Release rate constants are determined for each product and used to construct a quantitative model of the mechanism that accurately predicts the behavior of this complex system.
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