Product release during the first turnover of the ATP sulfurylase-GTPase

S. Sukal, Thomas S. Leyh

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

ATP sulfurylase, from Escherichia coli K-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 Pi 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·PPi·E·GDP·Pi and AMP·PPi·E·GDP, the rate constants for the release of Pi (4.2 s-1) and GDP (4.8 s-1) from these complexes, respectively, and the equilibrium constant for the enzyme-bound, β,γ-bond cleavage reaction: [AMP·PPi·E·GTP']/ [AMP·PPi· E·GDP·Pi] = 0.7. The data are fit, using global analysis, to obtain a complete kinetic and energetic description of this GTPase reaction.

Original languageEnglish (US)
Pages (from-to)15009-15016
Number of pages8
JournalBiochemistry
Volume40
Issue number49
DOIs
StatePublished - Dec 11 2001

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Sulfate Adenylyltransferase
GTP Phosphohydrolases
Chemical potential
Isomerization
Enzymes
Equilibrium constants
Guanosine Triphosphate
Escherichia coli
Sulfates
Hydrolysis
Rate constants
Assays
Experiments
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Product release during the first turnover of the ATP sulfurylase-GTPase. / Sukal, S.; Leyh, Thomas S.

In: Biochemistry, Vol. 40, No. 49, 11.12.2001, p. 15009-15016.

Research output: Contribution to journalArticle

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