ATP sulfurylase catalyzes and chemically links the hydrolysis of GTP and the synthesis of activated sulfate (APS). Like many GTPases, its GTPase activity is allosterically regulated, in this case, by APS-forming reactants and their analogues. Using these activators, we have been able to mimic many of the complexes that form in the native reaction, including an DAMP intermediate. The effects of each of these complexes on GTP hydrolysis are determined. The results of pre-steady-state and isotope trapping studies demonstrate that the binding of activator and substrate to the enzyme are near equilibrium and that the rate-determining step appears to be scission of the /?,y-bond of GTP. These properties of the system allow the energetic consequences of activator binding on the ground-and transition-state complexes to be evaluated. Activation occurs predominantly by transition-state stabilization, resulting in kcat increases.The values for kcat span a 180-fold range and vary with each activator. Km, or ground-state, effects are relatively small, -3-fold, and are uniform throughout the activator series. These studies provide an indepth view of the energetic interactions between the two active sites at each step of the APS-forming reaction.

ATP sulfurylase, isolated from Escherichia coli K-12, is a GTPase-target complex that catalyzes and links the energetics of GTP hydrolysis to the synthesis of activated sulfate (APS). When the GTP concentration is saturating and held fixed with a regenerating system, the APS reaction reaches a steady state in which its mass ratio is shifted (5.4 x 10(6))-fold toward the product by the hydrolysis of GTP. If GTP is not regenerated, the shift toward the product is transient, producing a pulse-shaped progress curve. The mechanistic basis of this transience is the subject of this paper. The product transient is caused by the binding of GDP to the enzyme which establishes a catalytic pathway that allows the chemical potential that had been transferred to the APS reaction to "leak" into the chemical milieu. The system leaks because the E.GDP complex catalyzes the uncoupled APS reaction. The addition of phosphate to the leaky GDP.E.APS.PPi complex converts it into the central Pi.GDP.E.APS.PPi complex which catalyzes the energy-transfer reaction. Thus, Pi binding directs the system through the coupled mechanism, "plugging" the leak. GMPPNP, which also causes a leak, is used to demonstrate that the mass ratio of the APS reaction can be "tuned" by adjusting flux through the coupled and uncoupled pathways. This energy-coupling mechanism provides a means for controlling the quantity of chemical potential transferred to the APS reaction. This versatile linkage might well be used to the cell's advantage to avoid the toxicity associated with an excess of activated sulfate.