Product release during the first turnover of the ATP sulfurylase-GTPase.
ATP sulfurylase, from Escherichia coli Kappa-12, is a GTPase target complex that catalyzes and couples the chemical potentials of two reactions: GTP hydrolysis and activated sulfate (APS) synthesis. Previous work suggested that the product release branch of the GTPase mechanism might include rate-determining release and/or isomerization step(s). Such steps are known to couple chemical potentials in other energy transducing systems. Rate-determining, product release step(s) were confirmed in the ATP sulfurylase-GTPase reaction by a burst of product in pre-steady-state, rapid-quench experiments. Classical rapid-quench experiments, which measure total product formation, do not allow the slow steps to be assigned to the release of a specific product, or to slow isomerization, because they do not distinguish solution-phase from enzyme-bound product. Assay systems that exclusively monitor solution-phase P(i) and GDP were used to obtain free product progress curves during the first turnover of ATP sulfurylase. Together, the free and total product data describe how the products partition between the enzyme surface and solution during the first turnover. In combination, the data provide the time dependence of the concentrations of specific product intermediates, AMP.PP(i).E.GDP.P(i) and AMP.PP(i).E.GDP, the rate constants for the release of P(i) (4.2 s(-1)) and GDP (4.8 s(-1)) from these complexes, respectively, and the equilibrium constant for the enzyme-bound, beta,gamma-bond cleavage reaction: [AMP.PP(i).E.GTP']/[AMP.PP(i).E.GDP.P(i)] = 0.7. The data are fit, using global analysis, to obtain a complete kinetic and energetic description of this GTPase reaction.
Sukal
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The Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461-1926, USA.
Leyh
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D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D051528:Guanylate Kinases; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D013430:Sulfate Adenylyltransferase / Q000378:metabolism*; D013997:Time Factors
D010710:Phosphates; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases; D013430:Sulfate Adenylyltransferase; D020558:GTP Phosphohydrolases