The role of enzyme isomerization in the native catalytic cycle of the ATP sulfurylase-GTPase system.
ATP sulfurylase, from E. coli Kappa-12, is a GTPase.target complex that conformationally couples the free energies of GTP hydrolysis and activated sulfate (adenosine 5'-phosphosulfate, or APS) synthesis. Energy coupling is achieved by an allosterically driven isomerization that switches on and off chemistry at specific points in the catalytic cycle. This coupling mechanism is derived from the results of model studies using analogue complexes that mimic 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]triphosphate) and mGTP [3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-triphosphate]. The 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 and quantitate the isomerization mechanism, and to show that the isomerization precedes and rate-limits both GTP hydrolysis and APS synthesis. These findings are incorporated into a model of the energy-coupling mechanism.
Wei
J
J
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461-1926, USA.
Liu
C
C
Leyh
T S
TS
D000250:Adenosine Phosphosulfate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000495:Allosteric Site; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006868:Hydrolysis; D007536:Isomerism; D007700:Kinetics; D013430:Sulfate Adenylyltransferase / Q000737:chemistry* / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism; D013816:Thermodynamics
D013431:Sulfates; D006165:Guanylyl Imidodiphosphate; D000250:Adenosine Phosphosulfate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D013430:Sulfate Adenylyltransferase; D020558:GTP Phosphohydrolases