The Energetic Linkage of GTP Hydrolysis and the Synthesis of Activated Sulfate

Chang-xian, Liu; Suo, Ya; Leyh, Thomas S.

doi:10.1021/bi00189a036

Cited by 37 publications

(43 citation statements)

References 31 publications

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“…3B). The apparent equilibrium constant for the APS-forming reaction in the presence of GTP is 0.23, which is comparable with the value of 0.059 determined using the E. coli system (32). The chemical potential of the hydrolysis reaction that is coupled to APS synthesis can be calculated from the difference in the potential of the APS-forming reaction in the presence and absence of GTP.…”

Section: Resultssupporting

confidence: 74%

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: Resultssupporting

confidence: 74%

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

Sun

Andreassi

Liu

et al. 2005

Journal of Biological Chemistry

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“…[12] For example, the elongation factor Tu (EF-Tu), which takes part in the synthesis of polypeptides in cells, forms a complex with Mg 2 , GTP, and aminoacyl-tRNA that interacts with the codon-programmed ribosome; this results in the binding of aminoacyl-tRNA to the ribosomal acceptor site, the hydrolysis of GTP, and the release of EF-Tu´MgǴ DP. [13] GTP hydrolysis is also essential for the insertion of nickel into hydrogenases [14] or the synthesis of activated sulfate, [15] which is an essential step in the metabolic assimilation of sulfur.…”

Section: Introductionmentioning

confidence: 99%

Stabilities and Isomeric Equilibria in Solutions of Monomeric Metal-Ion Complexes of Guanosine 5′-Triphosphate (GTP4−) and Inosine 5′-Triphosphate (ITP4−) in Comparison with Those of Adenosine 5′-Triphosphate (ATP4−)

et al. 2001

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Under experimental conditions in which the self-association of the purine-nucleoside 5'-triphosphates (PuNTPs) GTP and ITP is negligible, potentiometric pH titrations were carried out to determine the stabilities of the M(H;PuNTP) and M(PuNTP)2-complexes where M2+ = Mg2+, Ca2+, Sr2+. Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, or Cd2+ (I = 0.1 M, 25 degrees C). The stabilities of all M(GTP)2- and M(ITP)2- complexes are significantly larger than those of the corresponding complexes formed with pyrimidine-nucleoside 5'-triphosphates (PyNTPs), which had been determined previously under the same conditions. This increased complex stability is attributed, in agreement with previous 1H MNR shift studies, to the formation of macrochelates of the phosphate-coordinated metal ions with N7 of the purine residues. A similar enhanced stability (despite relatively large error limits) was observed for the M(H;PuNTP) complexes, in which H+ is bound to the terminal y-phosphate group, relative to the stability of the M(H;PyNTP)- species. The percentage of the macrochelated isomers in the M(GTP)2- and M(ITP)2- systems was quantified by employing the difference log KMM(PuNTP)-log KMM(PyNTP); the lowest and highest formation degrees of the macrochelates were observed for Mg(ITP)2- and Cu(GTP)2- with 17 +/- 11% and 97 +/- 1%, respectively. From previous studies of M(ATP)2- complexes, it is known that innersphere and outersphere macrochelates may form; that is, in the latter case a water molecule is between N7 and the phosphate-coordinated M2+. Similar conclusions are reached now by comparisons with earlier 1H MNR shift measurements, that is, that Mg(GTP)2- (21 +/- 11%), for example, exists largely in the form of an outersphere macrochelate and Zn(GTP)2- (68 +/- 4%) as an innersphere one. Generally, the overall percentage of macrochelate falls off for a given metal ion in the order M(GTP)2- > M(ITP)2- > M(ATP)2-; this is in accord with the decreasing basicity of N7 and the steric inhibition of the (C6)NH2 group in the adenine residue. Furthermore, although the absolute stability constants of the previously studied M(GMP), M(IMP), and M(AMP) complexes differ by about two to three log units from the present M(PuNTP)2- results, the formation degrees of the macrochelates are astonishingly similar for the two series of nucleotides for a given metal ion and purine-nucleobase residue. The conclusion that N7 of the guanine residue is an especially favored binding site for metal ions is also in accord with observations made for nucleic acids.

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“…The enzyme that performs this task, ATP sulfurylase (CysD), forms part of a heterodimer. The other component is a GTPase (CysN) that couples the hydrolysis of GTP to the sulfurylation of ATP, thereby providing energy to shift the equilibrium so as to favor the formation of the product (15,16). APS kinase (CysC) then converts APS to PAPS.…”

mentioning

confidence: 99%

5′-Adenosinephosphosulfate Lies at a Metabolic Branch Point in Mycobacteria

Williams

Senaratne

Mougous

et al. 2002

Journal of Biological Chemistry

124

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Bacterial sulfate assimilation pathways provide for activation of inorganic sulfur for the biosynthesis of cysteine and methionine, through either adenosine 5-phosphosulfate (APS) or 3-phosphoadenosine 5-phosphosulfate (PAPS) as intermediates. PAPS is also the substrate for sulfotransferases that produce sulfolipids, putative virulence factors, in Mycobacterium tuberculosis such as SL-1. In this report, genetic complementation using Escherichia coli mutant strains deficient in APS kinase and PAPS reductase was used to define the M. tuberculosis and Mycobacterium smegmatis CysH enzymes as APS reductases. Consequently, the sulfate assimilation pathway of M. tuberculosis proceeds from sulfate through APS, which is acted on by APS reductase in the first committed step toward cysteine and methionine. Thus, M. tuberculosis most likely produces PAPS for the sole use of this organism's sulfotransferases. Deletion of CysH from M. smegmatis afforded a cysteine and methionine auxotroph consistent with a metabolic branch point centered on APS. In addition, we have redefined the substrate specificity of the B. subtilis CysH, formerly designated a PAPS reductase, as an APS reductase, based on its ability to complement a mutant E. coli strain deficient in APS kinase. Together, these studies show that two conserved sequence motifs, CCXXRKXXPL and SXGCXXCT, found in the C termini of all APS reductases, but not in PAPS reductases, may be used to predict the substrate specificity of these enzymes. A functional domain of the M. tuberculosis CysC protein was cloned and expressed in E. coli, confirming the ability of this organism to make PAPS. The expression of recombinant M. tuberculosis APS kinase provides a means for the discovery of inhibitors of this enzyme and thus of the biosynthesis of SL-1.

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The Energetic Linkage of GTP Hydrolysis and the Synthesis of Activated Sulfate

Cited by 37 publications

References 31 publications

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

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

Stabilities and Isomeric Equilibria in Solutions of Monomeric Metal-Ion Complexes of Guanosine 5′-Triphosphate (GTP4−) and Inosine 5′-Triphosphate (ITP4−) in Comparison with Those of Adenosine 5′-Triphosphate (ATP4−)

5′-Adenosinephosphosulfate Lies at a Metabolic Branch Point in Mycobacteria

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