Allosteric Regulation of the ATP Sulfurylase Associated GTPase

Wang, Ruixiu; Chang-xian, Liu; Leyh, Thomas S.

doi:10.1021/bi00002a013

Cited by 16 publications

(32 citation statements)

References 30 publications

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“…The isomerization commits the chemistries to forward reaction and appears to bring residues at both active sites into their catalytic positions, from which chemistry occurs quickly (35). Together, the substrate analogues AMP and PP i can substitute for ATP and SO 4 in driving the isomerization and activating GTP hydrolysis (36).…”

Section: Resultsmentioning

confidence: 99%

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%

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

Sun

Andreassi

Liu

et al. 2005

Journal of Biological Chemistry

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“…A two-stage mixing experiment was designed to determine if exchange between the eEF1A-bound and free pools of GTP is fast compared with product formation (48). To perform this experiment, eEF1A is first mixed with a saturating concentration of [␥-…”

Section: Resultsmentioning

confidence: 99%

The Effect of F-actin on the Binding and Hydrolysis of Guanine Nucleotide by Dictyostelium Elongation Factor 1A

Edmonds¹,

Bell²,

Wyckoff³

et al. 1998

Journal of Biological Chemistry

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Indirect evidence implicates actin as a cofactor in eukaryotic protein synthesis. The present study directly examines the effects of F-actin on the biochemical properties of eukaryotic elongation factor 1A (eEF1A, formerly EF1␣), a major actin-binding protein. The basal mechanism of eEF1A alone is determined under physiological conditions with the critical finding that glycerol and guanine nucleotide are required to prevent protein aggregation and loss of enzymatic activity. The dissociation constants (K d ) for GDP and GTP are 2.5 M and 0.6 M, respectively, and the k cat of GTP hydrolysis is 1.0 ؋ 10 ؊3 s ؊1 . When eEF1A binds to F-actin, there is a 7-fold decrease in the affinity for guanine nucleotide and an increase of 35% in the rate of GTP hydrolysis. Based upon our results and the relevant cellular concentrations, the predominant form of cellular eEF1A is calculated to be GTP⅐eEF1A⅐F-actin. We conclude that Factin does not significantly modulate the basal enzymatic properties of eEF1A; however, actin may still influence protein synthesis by sequestering GTP⅐eEF1A away from interactions with its known translational ligands, e.g. aminoacyl-tRNA and ribosomes.Actin has been implicated as a co-factor in eukaryotic protein synthesis (reviewed in Refs. 1-3). Early cell ultrastructural studies revealed an association between components of the translational machinery and the actin cytoskeleton (4, 5), and functional studies showed that mRNAs associated with detergent-insoluble material, presumably the cytoskeleton, are translated more efficiently (6). Subsequently, it has been shown that virtually all of the protein components of translation associate with the cytoskeleton in situ (7-12); however, no direct effect of the cytoskeleton on the specific activity of any of these co-factors has been demonstrated.Eukaryotic elongation factor 1A (eEF1A, 1 formerly EF1␣) 2 is the most abundant G protein in cells, comprising 1-5% of total cell protein (14 -16). The principal role of eEF1A during protein synthesis is to bind aminoacyl-tRNA to the A-site of the ribosome by a GTP-dependent mechanism (17). Following the hydrolysis of GTP, aminoacyl-tRNA is incorporated into the growing polypeptide, and GDP⅐eEF1A is released from the ribosome. The exchange of bound GDP for free GTP regenerates the capacity of eEF1A to bind another aminoacyl-tRNA and to participate in further cycles of elongation. Therefore, the affinity of eEF1A for GDP and GTP and the kinetics of GTP hydrolysis are potential targets for ligands operating through eEF1A to regulate protein synthesis. For example, the rate of GDP/ GTP exchange by eEF1A in vitro is too slow to support the measured rate of polypeptide elongation in vivo; therefore, the need for a nucleotide exchange factor has been postulated (18,19).eEF1A was identified in a screen for actin binding proteins in Dictyostelium and was shown to bind both monomeric and filamentous actin (F-actin) in vitro and in situ (20 -23). Due to the striking similarity of eEF1A primary sequences among species, it...

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“…; this value, which is 11 % of k cat for the E. coli ATP sulfurylase (Wang et al, 1995), establishes a lower limit for the k cat of the GTPase-activated turnover of the CysD subunit in the M. tuberculosis complex. It should be noted that ATP sulfurylase activity in crude extracts of E. coli grown on rich media, which potently suppresses expression of the cys regulon, is below detectable limits (JonesMortimer et al, 1968).…”

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confidence: 85%

The Mycobacterium tuberculosis cysD and cysNC genes form a stress-induced operon that encodes a tri-functional sulfate-activating complex

et al. 2004

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The Mycobacterium tuberculosis cysD and cysNC genes form a stress-induced operon that encodes a tri-functional sulfate-activating complex Sulfur metabolism has been implicated in the virulence, antibiotic resistance and anti-oxidant defence of Mycobacterium tuberculosis. Despite its human disease relevance, sulfur metabolism in mycobacteria has not yet been fully characterized. ATP sulfurylase catalyses the synthesis of activated sulfate (adenosine 59-phosphosulfate, APS), the first step in the reductive assimilation of sulfate. Expression of the M. tuberculosis cysD gene, predicted to encode the adenylyl-transferase subunit of ATP sulfurylase, is upregulated by the bacilli inside its preferred host, the macrophage. This study demonstrates that cysD and cysNC orthologues exist in M. tuberculosis and constitute an operon whose expression is induced by sulfur limitation and repressed by the presence of cysteine, a major end-product of sulfur assimilation. The cysDNC genes are also induced upon exposure to oxidative stress, suggesting regulation of sulfur assimilation by M. tuberculosis in response to toxic oxidants. To ensure that the cysDNC operon encoded the activities predicted by its primary sequence, and to begin to characterize the products of the operon, they were expressed in Escherichia coli, purified to homogeneity, and tested for their catalytic activities. The CysD and CysNC proteins were shown to form a multifunctional enzyme complex that exhibits the three linked catalytic activities that constitute the sulfate activation pathway. Abbreviations: APS, adenosine 59-phosphosulfate; PAPS, 39-phosphoadenosine 59-phosphosulfate; PB, Proskauer and Beck.

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Allosteric Regulation of the ATP Sulfurylase Associated GTPase

Cited by 16 publications

References 30 publications

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

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

The Effect of F-actin on the Binding and Hydrolysis of Guanine Nucleotide by Dictyostelium Elongation Factor 1A

The Mycobacterium tuberculosis cysD and cysNC genes form a stress-induced operon that encodes a tri-functional sulfate-activating complex

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