ATP sulfurylase from Escherichia coli K12 catalyzes two, coupled reactions: the hydrolysis of GTP and the formation of activated sulfate (APS). At saturating levels of GTP, the initial rate of APS formation is stimulated 116-fold. The mechanism of this activation has been investigated using isotope trapping, mass spectrometry, and initial velocity kinetic techniques. In the presence of GTP, APS formation proceeds via nucleophilic attack of sulfate at the α-phosphoryl group of ATP. Isotope-trapping experiments demonstrate productive, random binding of ATP and GTP. ATP is hydrolyzed to yield AMP and PPi AMP production requires GTP and is suppressible by sulfate, suggesting GTP-dependent formation of an E*AMP intermediate in the synthesis of APS. Studies using the hydrolysis-resistant nucleotide analogues AMPCPP and GMPPNP demonstrate that GTP hydrolysis precedes scision of the α–β bond of ATP. Product inhibition studies indicate that PPi release occurs prior to the addition of sulfate and APS formation. These results are used to construct a proposed mechanism for the GTP-activated synthesis of APS.
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