GTPase-mediated activation of ATP sulfurylase.

Leyh, Thomas S.; Suo, Ya

doi:10.1016/s0021-9258(18)48528-6

Cited by 40 publications

(13 citation statements)

References 29 publications

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“…ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4), from E. coli K-12, catalyzes and couples the chemical potentials of GTP hydrolysis and the synthesis of activated sulfate (adenosine 5′-phosphosulfate, or APS), reactions 1 and 2 (11,12):…”

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confidence: 99%

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Conformational Change Rate-Limits GTP Hydrolysis: The Mechanism of the ATP Sulfurylase−GTPase

Jiang

Leyh

1998

Biochemistry

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The fluorescent GTP analogues 3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine 5'-(beta, gamma-imidotriphosphate) (mGMPPNP) and 3'-O-(N-methylanthraniloyl)-2'-deoxy-GTP (mGTP) were used to demonstrate that an enzyme isomerization precedes and rate-limits beta,gamma-bond cleavage in the catalytic cycle of the ATP sulfurylase-GTPase, from E. coli K-12. The binding of mGMPPNP to the E.AMP.PPi complex of ATP sulfurylase is biphasic, indicating that an isomerization occurs in the binding reaction. The isomerization mechanism was assigned based on the results of the enzyme concentration dependence of the observed rate constants, kobs, for both phases of the binding reaction, and sequential-mixing, nucleotide release experiments. The isomerization occurs after, and is driven by, the addition of mGMPPNP. Values were determined for each of the rate constants associated with the two-step kinetic model used in the interpretation of the results. A comparison of the enzyme concentration dependence of kobs for the hydrolysis and binding reactions reveals that the rate constants for the corresponding steps of these two reactions are extremely similar. The virtually identical rate constants for isomerization and beta, gamma-bond scission strongly suggest that isomerization rate-limits bond breaking. The implications of these finding for GTPase/target interactions and the mechanism of energetic linkage in the ATP sulfurylase system are discussed.

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confidence: 99%

“…ATP sulfurylase conformationally couples the chemical potential of GTP hydrolysis to that of activated sulfate synthesis (11,12). Conformational coupling is achieved through allosterically driven structural changes that link events that occur in the separate reactions to one another.…”

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confidence: 99%

Conformational Change Rate-Limits GTP Hydrolysis: The Mechanism of the ATP Sulfurylase−GTPase

Jiang

Leyh

1998

Biochemistry

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“…The adenosine 5′-phosphosulfate (APS) formed in this reaction contains the mixed phosphoric-sulfuric acid anhydride bond that is the hallmark of activated sulfate. ∆G°′ for the hydrolysis of this bond (-19 kcal/mol (2)) is considerably higher than that for the R,β-bond of ATP (-10.7 kcal/mol) which is cleaved in the transfer (3). Consequently, the formation of APS and PP i is extremely unfavorable; the equilibrium constant for reaction 1 is 1.1 × 10 -8 at pH 8.0 (4).…”

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confidence: 92%

Regulating Energy Transfer in the ATP Sulfurylase−GTPase System

et al. 1998

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ATP sulfurylase, isolated from Escherichia coli K-12, is a GTPase-target complex that catalyzes and links the energetics of GTP hydrolysis to the synthesis of activated sulfate (APS). When the GTP concentration is saturating and held fixed with a regenerating system, the APS reaction reaches a steady state in which its mass ratio is shifted (5.4 x 10(6))-fold toward the product by the hydrolysis of GTP. If GTP is not regenerated, the shift toward the product is transient, producing a pulse-shaped progress curve. The mechanistic basis of this transience is the subject of this paper. The product transient is caused by the binding of GDP to the enzyme which establishes a catalytic pathway that allows the chemical potential that had been transferred to the APS reaction to "leak" into the chemical milieu. The system leaks because the E.GDP complex catalyzes the uncoupled APS reaction. The addition of phosphate to the leaky GDP.E.APS.PPi complex converts it into the central Pi.GDP.E.APS.PPi complex which catalyzes the energy-transfer reaction. Thus, Pi binding directs the system through the coupled mechanism, "plugging" the leak. GMPPNP, which also causes a leak, is used to demonstrate that the mass ratio of the APS reaction can be "tuned" by adjusting flux through the coupled and uncoupled pathways. This energy-coupling mechanism provides a means for controlling the quantity of chemical potential transferred to the APS reaction. This versatile linkage might well be used to the cell's advantage to avoid the toxicity associated with an excess of activated sulfate.

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“…Thus, despite the fact that APS synthesis is coupled to cleavage of the ATP α,β-bond (), the equilibrium constant for APS formation is remarkably unfavorable [1.1 × 10 -8 , pH 8.0 ( , )]. This formidable energetic barrier is overcome in E. coli (), and other gram-negative bacteria (), by coupling the free energies of GTP hydrolysis (reaction 2) and APS synthesis ( , ): These two reactions are catalyzed and energetically linked by the enzyme ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) from E. coli . This enzyme is a tetramer of heterodimers.…”

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confidence: 99%

“…Each heterodimer is composed of a CysD (35 kDa) and a CysN (53 kDa) subunit, which catalyze APS synthesis and GTP hydrolysis, respectively ( , ). ATP sulfurylase is a rare example of a GTPase·target complex in which structural changes in the catalytic cycle couple the free energy of GTP hydrolysis (reaction 2) to a second, small-molecule reaction ( , ).…”

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The Role of Enzyme Isomerization in the Native Catalytic Cycle of the ATP Sulfurylase−GTPase System

2000

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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.

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GTPase-mediated activation of ATP sulfurylase.

Cited by 40 publications

References 29 publications

Conformational Change Rate-Limits GTP Hydrolysis: The Mechanism of the ATP Sulfurylase−GTPase

Conformational Change Rate-Limits GTP Hydrolysis: The Mechanism of the ATP Sulfurylase−GTPase

Regulating Energy Transfer in the ATP Sulfurylase−GTPase System

The Role of Enzyme Isomerization in the Native Catalytic Cycle of the ATP Sulfurylase−GTPase System

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