Structural and functional analysis of a truncated form of Saccharomyces cerevisiae ATP sulfurylase: C-terminal domain essential for oligomer formation but not for activity.

ATP sulfurylase catalyzes the first step in the activation of sulfate by transferring the adenylyl-moiety (AMP approximately ) of ATP to sulfate to form adenosine 5'-phosphosulfate (APS) and pyrophosphate (PP(i)). Subsequently, APS kinase mediates transfer of the gamma-phosphoryl group of ATP to APS to form 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and ADP. The recently determined crystal structure of yeast ATP sulfurylase suggests that its C-terminal domain is structurally quite independent from the other domains, and not essential for catalytic activity. It seems, however, to dictate the oligomerization state of the protein. Here we show that truncation of this domain results in a monomeric enzyme with slightly enhanced catalytic efficiency. Structural alignment of the C-terminal domain indicated that it is extremely similar in its fold to APS kinase although not catalytically competent. While carrying out these structural and functional studies a surface groove was noted. Careful inspection and modeling revealed that the groove is sufficiently deep and wide, as well as properly positioned, to act as a substrate channel between the ATP sulfurylase and APS kinase-like domains of the enzyme.

Product release during the first turnover of the ATP sulfurylase-GTPase.

ATP sulfurylase, from Escherichia coli Kappa-12, is a GTPase target complex that catalyzes and couples the chemical potentials of two reactions: GTP hydrolysis and activated sulfate (APS) synthesis. Previous work suggested that the product release branch of the GTPase mechanism might include rate-determining release and/or isomerization step(s). Such steps are known to couple chemical potentials in other energy transducing systems. Rate-determining, product release step(s) were confirmed in the ATP sulfurylase-GTPase reaction by a burst of product in pre-steady-state, rapid-quench experiments. Classical rapid-quench experiments, which measure total product formation, do not allow the slow steps to be assigned to the release of a specific product, or to slow isomerization, because they do not distinguish solution-phase from enzyme-bound product. Assay systems that exclusively monitor solution-phase P(i) and GDP were used to obtain free product progress curves during the first turnover of ATP sulfurylase. Together, the free and total product data describe how the products partition between the enzyme surface and solution during the first turnover. In combination, the data provide the time dependence of the concentrations of specific product intermediates, AMP.PP(i).E.GDP.P(i) and AMP.PP(i).E.GDP, the rate constants for the release of P(i) (4.2 s(-1)) and GDP (4.8 s(-1)) from these complexes, respectively, and the equilibrium constant for the enzyme-bound, beta,gamma-bond cleavage reaction: [AMP.PP(i).E.GTP']/[AMP.PP(i).E.GDP.P(i)] = 0.7. The data are fit, using global analysis, to obtain a complete kinetic and energetic description of this GTPase reaction.

Vibrational structure of GDP and GTP bound to RAS: an isotope-edited FTIR study.

A complete vibrational description of the bonding of a ligand to a protein requires the assignment of both symmetric and antisymmetric vibrational modes. The symmetric modes of isotopically enriched enzyme-bound ligands can be obtained by Raman difference spectroscopy, but until now, the antisymmetric modes, which require IR difference spectroscopy, have not been generally accessible. We have developed the methodology needed to perform IR difference spectroscopy, assign the antisymmetric modes, and accurately describe bonding. The method is used to assess the bonding changes that occur as Mg.GDP and Mg.GTP move from solution into the active site of RAS. Binding to RAS opens the nonbridging, O--P--O angle of the gamma-phosphate of GTP by 2.7 degrees, yet the angular freedom (dispersion of the O--P--O angle) of the gamma-phosphate is comparable to that in solution. In contrast, the motion of the beta-phosphate of GDP is highly restricted, suggesting that it positions the gamma-phosphate for nucleophilic attack. The beta,gamma-bridging O-P bond of bound GTP is slightly weakened, being lengthened by 0.005 A in the active site, corresponding to a bond order decrease of 0.012 valence unit (vu). The observed binding changes are consistent with a RAS-mediated hydrolysis mechanism that parallels that for solution hydrolysis.

gamma-phosphate protonation and pH-dependent unfolding of the Ras.GTP.Mg2+ complex: a vibrational spectroscopy study.

The interdependence of GTP hydrolysis and the second messenger functions of virtually all GTPases has stimulated intensive study of the chemical mechanism of the hydrolysis. Despite numerous mutagenesis studies, the presumed general base, whose role is to activate hydrolysis by abstracting a proton from the nucleophilic water, has not been identified. Recent theoretical and experimental work suggest that the gamma-phosphate of GTP could be the general base. The current study investigates this possibility by studying the pH dependence of the vibrational spectrum of the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes. Isotope-edited IR studies of the Ras.GTP.Mg(2+) complex show that GTP remains bound to Ras at pH as low as 2.0 and that the gamma-phosphate is not protonated at pH > or = 3.3, indicating that the active site decreases the gamma-phosphate pK(a) by at least 1.1 pK(a) units compared with solution. Amide I studies show that the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes partially unfold in what appear to be two transitions. The first occurs in the pH range 5.4-2.6 and is readily reversible. Differences in the pH-unfolding midpoints for the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes (3.7 and 4.8, respectively) reveal that the enzyme-gamma-phosphoryl interactions stabilize the structure. The second transition, pH 2.6-1.7, is not readily reversed. The pH-dependent unfolding of the Ras.GTP.Mg(2+) complex provides an alternative interpretation of the data that had been used to support the gamma-phosphate mechanism, thereby raising the issue of whether this mechanism is operative in GTPase-catalyzed GTP hydrolysis reactions.

The role of enzyme isomerization in the native catalytic cycle of the ATP sulfurylase-GTPase system.

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.

Isomerization couples chemistry in the ATP sulfurylase-GTPase system.

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.

Conformational change rate-limits GTP hydrolysis: the mechanism of the ATP sulfurylase-GTPase.

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.

Sulfuryl transfer: the catalytic mechanism of human estrogen sulfotransferase.

Estrogen sulfotransferase (EST) catalyzes the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to 17beta-estradiol (E2). The sulfation of E2 prevents it from binding to, and thereby activating, the estrogen receptor. The regulation of EST appears to be causally linked to tumorigenesis in the breast and endometrium. In this study, recombinant human EST is characterized, and the catalytic mechanism of the transfer reaction is investigated in ligand binding and initial rate experiments. The native enzyme is a dimer of 35-kDa subunits. The apparent equilibrium constant for transfer to E2 is (4.5 +/- 0.2) x 10(3) at pH 6.3 and T = 25 +/- 2 degrees C. Initial rate studies provide the kinetic constants for the reaction and suggest a sequential mechanism. E2 is a partial substrate inhibitor (Ki = 80 +/- 5 nM). The binding of two E2 per EST subunit suggests that the partial inhibition occurs through binding at an allosteric site. In addition to providing the dissociation constants for the ligand-enzyme complexes, binding studies demonstrate that each substrate binds independently to the enzyme and that both the E.PAP.E2S and E.PAP.E2 dead-end complexes form. These results strongly suggest a Random Bi Bi mechanism with two dead-end complexes.

The effect of F-actin on the binding and hydrolysis of guanine nucleotide by Dictyostelium elongation factor 1A.

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 EF1alpha), 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 (Kd) for GDP and GTP are 2.5 microM and 0.6 microM, respectively, and the kcat of GTP hydrolysis is 1.0 x 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 GTP.eEF1A.F-actin. We conclude that F-actin does not significantly modulate the basal enzymatic properties of eEF1A; however, actin may still influence protein synthesis by sequestering GTP.eEF1A away from interactions with its known translational ligands, e.g. aminoacyl-tRNA and ribosomes.

Regulating energy transfer in the ATP sulfurylase-GTPase system.

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.

Altering the reaction coordinate of the ATP sulfurylase-GTPase reaction.

ATP sulfurylase, isolated from Escherichia coli K-12, catalyzes and couples two reactions: the hydrolysis of GTP and the synthesis of APS (adenosine 5'-phosphosulfate). Its GTPase activity is regulated in response to ligand binding at the APS-forming active site. In particular, AMP mimics an intermediate-like form of the enzyme that increases the k(cat) for GTP hydrolysis 180-fold. Using equilibrium and pre-steady-state methods, we have determined the relative Gibbs energies for many of the ground and transition states in the GTPase catalytic cycle, in the presence and absence of AMP. GTP and AMP energetically interact throughout the substrate branch of the reaction coordinate; however, once bond breaking occurs, communication between nucleotides ceases. Stopped-flow experiments, using the fluorescent nucleotides 2'-deoxy-mant-GTP and -GDP, indicate that the binding of AMP fosters a conformation of the enzyme that hinders the addition of 2'-deoxy-mant-GTP into the active site without affecting its escaping tendency. These results explain the effects of AMP on the equilibrium binding of the 2'-deoxy-mant-GTP. The second-order rate constants for the binding of 2'-deoxy-mant-GTP or -GDP, approximately 1 x 10(-6) M(-1) s(-1), are 2-3 orders of magnitude less than expected for simple diffusion models, and the binding progress curves appear biphasic. These findings suggest the presence of an intermediate(s) in the binding reactions. The Gibbs energy changes that occur in the reaction coordinate upon binding of AMP clearly show that the catalytic effect of AMP is due primarily to its -3.1 kcal/mol stabilization of the rate-limiting transition state.

Allosteric regulation of the ATP sulfurylase associated GTPase.

ATP sulfurylase catalyzes and chemically links the hydrolysis of GTP and the synthesis of activated sulfate (APS). Like many GTPases, its GTPase activity is allosterically regulated, in this case, by APS-forming reactants and their analogues. Using these activators, we have been able to mimic many of the complexes that form in the native reaction, including an E.AMP intermediate. The effects of each of these complexes on GTP hydrolysis are determined. The results of pre-steady-state and isotope trapping studies demonstrate that the binding of activator and substrate to the enzyme are near equilibrium and that the rate-determining step appears to be scission of the beta, gamma-bond of GTP. These properties of the system allow the energetic consequences of activator binding on the ground- and transition-state complexes to be evaluated. Activation occurs predominantly by transition-state stabilization, resulting in kcat increases. The values for kcat span a 180-fold range and vary with each activator. Km, or ground-state, effects are relatively small, approximately 3-fold, and are uniform throughout the activator series. These studies provide an in-depth view of the energetic interactions between the two active sites at each step of the APS-forming reaction.

Rhizobium meliloti NodP and NodQ form a multifunctional sulfate-activating complex requiring GTP for activity.

The nodulation genes nodP and nodQ are required for production of Rhizobium meliloti nodulation (Nod) factors. These sulfated oligosaccharides act as morphogenic signals to alfalfa, the symbiotic host of R. meliloti. In previous work, we have shown that nodP and nodQ encode ATP sulfurylase, which catalyzes the formation of APS (adenosine 5'-phosphosulfate) and PPi. In the subsequent metabolic reaction, APS is converted to PAPS (3'-phosphoadenosine 5'-phosphosulfate) by APS kinase. In Escherichia coli, cysD and cysN encode ATP sulfurylase; cysC encodes APS kinase. Here, we present genetic, enzymatic, and sequence similarity data demonstrating that nodP and nodQ encode both ATP sulfurylase and APS kinase activities and that these enzymes associate into a multifunctional protein complex which we designate the sulfate activation complex. We have previously described the presence of a putative GTP-binding site in the nodQ sequence. The present report also demonstrates that GTP enhances the rate of PAPS synthesis from ATP and sulfate (SO4(2-)) by NodP and NodQ expressed in E. coli. Thus, GTP is implicated as a metabolic requirement for synthesis of the R. meliloti Nod factors.

The energetic linkage of GTP hydrolysis and the synthesis of activated sulfate.

ATP sulfurylase, from Escherichia coli K-12, catalyzes both the hydrolysis of GTP and the synthesis of activated sulfate (APS). This paper describes the energetic linkage of these reactions and the events that couple them. Steady-state and single-turnover experiments suggest that the binding of GTP inhibits APS production and that the hydrolysis of GTP is required to generate the enzyme form(s) that produces APS. It is this progression from the inhibitory, E-GTP, to the productive, E-GDP, complexes in the cycle of APS synthesis that energetically links these two reactions. This model stands in contrast to other GTPase/target systems in which the binding of GTP alone is sufficient to catalyze multiple turnovers of the target reaction. The stoichiometry of GTP hydrolysis to APS synthesis is 1:1, and equilibrium measurements show that -9.1 kcal/mol, produced by the hydrolysis of GTP, is used to thermodynamically drive production of APS and PPi. These findings establish the mechanism of energy transfer in this novel GTPase/target system, and substantially alter our understanding of the energetics of sulfate activation, an essential step in the metabolic assimilation of sulfur.

GTPase activation of ATP sulfurylase: the mechanism.

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 alpha-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 alpha-beta 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.

The physical biochemistry and molecular genetics of sulfate activation.

This article is an overview of current research in the area of sulfate activation. Emphasis is placed on presenting unresolved issues in an appropriate context for critical evaluation by the reader. The energetics of sulfate activation is reevaluated in light of recent findings that demonstrate that the synthesis of activated sulfate is thermodynamically driven by GTP hydrolysis. The structural and functional bases of this GTPase activation are discussed in detail. The bonding and hydrolysis of the high-energy, phosphoric-sulfuric acid anhydride bond of activated sulfate are presented along with an analysis of the importance of the divalent cation and pyrophosphate protonation in the equilibria governing activated sulfate formation. The molecular genetics of sulfate assimilation in prokaryotes is reviewed with an emphasis on the regulation of the pathway. Recent discoveries connecting sulfate activation to plant/microbe symbiogenesis are presented, as are several examples of the importance of activated sulfate in human metabolism and disease.

The DNA sequence of the sulfate activation locus from Escherichia coli K-12.

The DNA sequence of the sulfate activation locus from Escherichia coli K-12 has been determined. The sequence includes the structural genes encoding the enzymes ATP sulfurylase (cysD and cysN) and APS kinase (cysC) which catalyze the synthesis of activated sulfate. These are the only genes known to reside in the sulfate activation operon. Consensus elements of the operon promoter were identified, and the start codons and open reading frames of the Cys polypeptides were determined. During this work, another gene, iap, was partially sequenced and mapped. The activity of ATP sulfurylase is stimulated by an intrinsic GTPase. Comparison of the primary sequences of CysN and Ef-Tu revealed that CysN has conserved many of the residues integral to the three-dimensional structure important for guanine nucleotide binding in Ef-Tu and RAS. nodP and nodQ, from Rhizobium meliloti, are essential for nodulation in leguminous plants. The Cys and Nod proteins are remarkably similar. NodP appears to be the smaller subunit of ATP sulfurylase. NodQ encodes homologues of both CysN and CysC; thus, these enzymes may be covalently associated in R. meliloti. The consensus GTP-binding sequences of NodQ and CysN are identical suggesting that NodQ encodes a regulatory GTPase.

GTPase-mediated activation of ATP sulfurylase.

GTP stimulates the synthesis of APS (adenosine 5'-phosphosulfate) by the enzyme ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) via a GTPase mechanism. The activation of the enzyme, purified from Escherichia coli, is titratable with GTP. The initial rate of APS formation is increased 116-fold at a saturating concentration of GTP. The enzyme exhibits a GTPase activity that is stimulated by ATP and further enhanced by SO4; however, SO4 alone does not significantly stimulate GTP hydrolysis. The larger subunit of ATP sulfurylase, encoded by cysN, contains a GTP-binding consensus sequence common to other known GTP-binding proteins. This is the first evidence that the sulfate activation pathway is a metabolic target for regulation by a GTPase.

cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth.

The initial steps in assimilation of sulfate during cysteine biosynthesis entail sulfate uptake and sulfate activation by formation of adenosine 5'-phosphosulfate, conversion to 3'-phosphoadenosine 5'-phosphosulfate, and reduction to sulfite. Mutations in a previously uncharacterized Escherichia coli gene, cysQ, which resulted in a requirement for sulfite or cysteine, were obtained by in vivo insertion of transposons Tn5tac1 and Tn5supF and by in vitro insertion of resistance gene cassettes. cysQ is at chromosomal position 95.7 min (kb 4517 to 4518) and is transcribed divergently from the adjacent cpdB gene. A Tn5tac1 insertion just inside the 3' end of cysQ, with its isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter pointed toward the cysQ promoter, resulted in auxotrophy only when isopropyl-beta-D-thiogalactopyranoside was present; this conditional phenotype was ascribed to collision between converging RNA polymerases or interaction between complementary antisense and cysQ mRNAs. The auxotrophy caused by cysQ null mutations was leaky in some but not all E. coli strains and could be compensated by mutations in unlinked genes. cysQ mutants were prototrophic during anaerobic growth. Mutations in cysQ did not affect the rate of sulfate uptake or the activities of ATP sulfurylase and its protein activator, which together catalyze adenosine 5'-phosphosulfate synthesis. Some mutations that compensated for cysQ null alleles resulted in sulfate transport defects. cysQ is identical to a gene called amtA, which had been thought to be needed for ammonium transport. Computer analyses, detailed elsewhere, revealed significant amino acid sequence homology between cysQ and suhB of E. coli and the gene for mammalian inositol monophosphatase. Previous work had suggested that 3'-phosphoadenoside 5'-phosphosulfate is toxic if allowed to accumulate, and we propose that CysQ helps control the pool of 3'-phosphoadenoside 5'-phosphosulfate, or its use in sulfite synthesis.