The role of enzyme isomerization in the native catalytic cycle of the ATP sulfurylase-GTPase system

ATP sulfurylase, from E. coli K-12, is a GTPase target complex that conform ationally couples the free energies of GTP hydrolysis and activated sulfate (adenosine S-phosphosulfate, or APS) synthesis. Energy coupling is achieve d by an allosterically driven isomerization that switches on and off chemis try at specific points in the catalytic cycle. This coupling mechanism is d erived from the results of model studies using analogue complexes that mimi c different stages of the native catalytic cycle. The current investigation extends the analogue studies to the native catalytic cycle. Isomerization is monitored using the fluorescent, guanine nucleotide analogues mGMPPNP (3 '-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'- [beta,gamma-imido]triphosp hate) and mGTP [3'-O-(N-methylanthraniloyl)-2'-deoxygua 5'-triphosphate]. T he isomerization is shown to be initiated by an allosteric interaction that requires the simultaneous occupancy of all three substrate-binding sites. Stopped-flow fluorescence and single-turnover studies were used to define a nd quantitate the isomerization mechanism, and to show that the isomerizati on precedes and rate-limits both GTP hydrolysis and APS synthesis. These fi ndings are incorporated into a model of the energy-coupling mechanism.