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

Jiang, Wei; Leyh, Thomas S.

doi:10.1021/bi9817461

Cited by 15 publications

(30 citation statements)

References 29 publications

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“…The Isomerization of SAC-The mechanism of ATP sulfurylase from E. coli includes an isomerization that is driven by allosteric interactions between ligands at the GTPase and adenylyl-transferase active sites (16,34). The isomerization, which precedes and partially rate-limits both GTP hydrolysis and APS synthesis, is a central energy-coupling step in the mechanism (34,35).…”

Section: Resultsmentioning

confidence: 99%

“…ATP was regenerated from ADP using pyruvate kinase, and the reactions were continuously monitored at 339 nm by coupling the regeneration of ATP to the oxidation of NADH using lactate dehydrogenase. The assay conditions were as follows: SAC (0.05 M), APS (64, 94, 177, and 1600 nM), ATP (16,24,45, and 400 M), pyruvate kinase (10 units/ml), lactate dehydrogenase (10 units/ml), HAL2 nucleotidase (0.5 unit/ml), Hepes (50 mM, pH/K ϩ ϭ 8.0), PEP (1.0 mM), NADH (0.25 mM), MgCl 2 (2.0 mM), and T ϭ 25 Ϯ 2°C.…”

Section: Enzchekmentioning

confidence: 99%

“…PAPS was purchased from Prof. S. Singer (University of Dayton, Dayton, OH). Recombinant E. coli ATP sulfurylase was expressed and purified as described previously (15,16). The yeast HAL2 (PAPS nucleotidase) bacterial expression plasmid, pETHAL2, was a generous gift from Prof. John D. York of Duke University Medical Center.…”

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confidence: 99%

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The Trifunctional Sulfate-activating Complex (SAC) of Mycobacterium tuberculosis

Sun

Andreassi

Liu

et al. 2005

Journal of Biological Chemistry

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The sulfate activation pathway is essential for the assimilation of sulfate and, in many bacteria, is comprised of three reactions: the synthesis of adenosine 5-phosphosulfate (APS), the hydrolysis of GTP, and the 3-phosphorylation of APS to produce 3-phosphoadenosine 5-phosphosulfate (PAPS), whose sulfuryl group is reduced or transferred to other metabolites. The entire sulfate activation pathway is organized into a single complex in Mycobacterium tuberculosis. Although present in many bacteria, these tripartite complexes have not been studied in detail. Initial rate characterization of the mycobacterial system reveals that it is poised for extremely efficient throughput: at saturating ATP, PAPS synthesis is 5800 times more efficient than APS synthesis. The APS kinase domain of the complex does not appear to form the covalent E⅐P intermediate observed in the closely related APS kinase from Escherichia coli. The stoichiometry of GTP hydrolysis and APS synthesis is 1:1, and the APS synthesis reaction is driven 1.1 ؋ 10 6 -fold further during GTP hydrolysis; the system harnesses the full chemical potential of the hydrolysis reaction to the synthesis of APS. A key energycoupling step in the mechanism is a ligand-induced isomerization that enhances the affinity of GTP and commits APS synthesis and GTP hydrolysis to the completion of the catalytic cycle. Ligand-induced increases in guanine nucleotide affinity observed in the mycobacterial system suggest that it too undergoes the energycoupling isomerization.The sulfate activation pathway in Mycobacterium tuberculosis is organized into a single complex that consists of three catalytic activities: an adenylyl-transferase (ATP sulfurylase), encoded by cysD, that catalyzes nucleophilic attack of sulfate at the ␣-phosphorous of ATP to produce adenosine 5Ј-phosphosulfate (APS); 1 a GTPase, encoded by cysN (a member of the EF-Tu family) (1, 2), whose activity is linked to the kinetics and energetics of the ATP sulfurylase reaction; and APS kinase, located at the C terminus of the cysN subunit, that phosphorylates APS at the 3Ј-hydroxyl to produce 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) (Reactions 1-3, respectively).The M. tuberculosis and Escherichia coli cysD and cysN sequences share considerable similarity; however, the E. coli APS kinase is expressed as a separate polypeptide, rather than a CysN domain. The organism-dependent fusion of the early cysteine biosynthetic enzymes is particularly interesting, given that the E. coli

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Section: Resultsmentioning

confidence: 99%

Section: Enzchekmentioning

confidence: 99%

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The Trifunctional Sulfate-activating Complex (SAC) of Mycobacterium tuberculosis

Sun

Andreassi

Liu

et al. 2005

Journal of Biological Chemistry

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“…In the sequential mechanism, the intermediate collapses to carbamate with phosphate as the leaving group, and at ATP C , the carbamate reacts with ATP to yield ADP and CP. In the switch mechanism, the intermediate collapses directly to CP on domain N, with water as the leaving group, and with domain C acting as an ATP‐driven molecular switch that allows the energetically unfavorable reaction to proceed on domain N. This scheme is analogous to mechanisms proposed for nicotinate phosphoribosyltransferase (Vinitsky and Grubmeyer 1993) and ATP sulfurylase (Wei and Leyh 1998).…”

mentioning

confidence: 85%

Nucleotide recognition in the ATP‐grasp protein carbamoyl phosphate synthetase

Kothe

Powers‐Lee

2004

Protein Science

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Synthesis of carbamoyl phosphate by carbamoyl phosphate synthetase (CPS) requires the coordinated utilization of two molecules of ATP per reaction cycle on duplicated nucleotide-binding sites (N and C). To clarify the contributions of sites N and C to the overall reaction, we carried out site-directed mutagenesis aimed at changing the substrate specificity of either of the two sites from ATP to GTP. Mutant design was based in part on an analysis of the nucleotide-binding sites of succinyl-CoA synthetases, which share membership in the ATP-grasp family with CPS and occur as GTP-and ATP-specific isoforms. We constructed and analyzed Escherichia coli CPS single mutations A144Q, D207A, D207N, S209A, I211S, P690Q, D753A, D753N, and F755A, as well as combinations thereof. All of the mutants retained ATP specificity, arguing for a lack of plasticity of the ATP sites of CPS with respect to nucleotide recognition. GTP-specific ATP-grasp proteins appear to accommodate this substrate by a displacement of the base relative to the ATP-bound state, an interaction that is precluded by the architecture of the potassium-binding loop in CPS. Analysis of the ATP-dependent kinetic parameters revealed that mutation of several residues conserved in ATP-grasp proteins and CPSs had surprisingly small effects, whereas constructs containing either A144Q or P690Q exerted the strongest effects on ATP utilization. We propose that these mutations affect proper movement of the lids covering the active sites of CPS, and interfere with access of substrate.

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“…The current model for energetic coupling in ATP sulfurylase suggests that the arrival of the APS-forming reaction at, or near, the E*AMP‚PP i intermediate that occurs in the catalytic cycle initiates an isomerization of the enzyme that accelerates GTP hydrolysis (18). This model is considerably expanded in this paper.…”

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confidence: 88%

Isomerization Couples Chemistry in the ATP Sulfurylase-GTPase System

Jiang

Leyh

1999

Biochemistry

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ATP sulfurylase catalyzes and couples the free energies of two reactions: GTP hydrolysis and the synthesis of activated sulfate, or APS. The GTPase active site undergoes changes during its catalytic cycle that are driven by events that occur at the APS-forming active site, which is located in a separate subunit. GTP responds to its changing environment by moving along its reaction path. The response, which may change the affinity or reactivity of GTP, can, in turn, produce alterations at the APS active site that drive APS synthesis. The resulting stepwise progression of the two reactions couples their free energies. The mechanism of ATP sulfurylase involves an enzyme isomerization that precedes and rate limits cleavage of the beta,gamma-bond of GTP. These fluorescence studies demonstrate that the isomerization is controlled by the binding of activators that drive ATP sulfurylase into forms that mimic different stages of the APS reaction. Only certain activators elicit the isomerization, suggesting that the APS reaction must proceed to a specific point in the catalytic cycle before the conformational "switch" that controls GTP hydrolysis is thrown. The isomerization is shown to require occupancy of the gamma-phosphate subsite of the GTP binding pocket. This requirement establishes that the isomerization results in a change in the interaction between the enzyme and the gamma-phosphate of GTP that emerges in the catalytic cycle during the transition from the nonisomerized to the isomerized E.GTP complex. The newly formed contact(s) appears to carry into the bond-breaking transition state, and to be essential for the enhanced affinity and reactivity of the nucleotide.

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

Cited by 15 publications

References 29 publications

The Trifunctional Sulfate-activating Complex (SAC) of Mycobacterium tuberculosis

The Trifunctional Sulfate-activating Complex (SAC) of Mycobacterium tuberculosis

Nucleotide recognition in the ATP‐grasp protein carbamoyl phosphate synthetase

Isomerization Couples Chemistry in the ATP Sulfurylase-GTPase System

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