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.     

Adenine nucleotide-binding sites on beef heart F1-ATPase. Conditions that affect occupancy of catalytic and noncatalytic sites

Beef heart mitochondrial F1 contains a total of six adenine nucleotide-binding sites including at least two different types of sites. Three "exchangeable" sites exchange rapidly during hydrolysis of MgATP, whereas three "nonexchangeable" sites do not (Cross, R. L. and Nalin, C. M. (1982) J. Biol. Chem. 257, 2874-2881). When F1 that has been stored as a suspension in (NH4)2SO4/ATP/EDTA/sucrose/Tris, pH 8.0, is pelleted, rinsed with (NH4)2SO4, dissolved, and desalted, it retains three bound adenine nucleotides. We find that two of these endogenous nucleotides are bound at nonexchangeable sites and one at an exchangeable site. The vacant nonexchangeable site is highly specific for adenine nucleotide and is rapidly filled by ADP upon addition of ADP or during ATP hydrolysis. ADP bound at this site can be removed by reprecipitating the enzyme with (NH4)2SO4. The single nucleotide retained by desalted F1 at an exchangeable site is displaced during hydrolysis of ATP, GTP, or ITP. The binding of PPi at two sites on the enzyme also promotes its dissociation. Neither procedure affects retention of nucleotide at the nonexchangeable sites. These observations, combined with the finding that PPi is much more easily removed from exchangeable sites than ADP, have led to the development of a procedure for preparing F1 with uniform and well-defined nucleotide site occupancy. This involves sequential exposure to MgATP, PPi, and high concentrations of Pi. Unbound ligand is removed between each step. The resulting enzyme, F1[3,0], has three occupied nonexchangeable sites and three vacant exchangeable sites. Evidence that nonexchangeable and exchangeable sites represent noncatalytic and catalytic sites, respectively, suggest that this form of the enzyme will prove useful in numerous applications, including transient kinetic measurements and affinity labeling of active site residues.     

Kinetic characterization of the GTPase activity of phage lambda terminase: evidence for communication between the two "NTPase" catalytic sites of the enzyme.

The terminase enzyme from bacteriophage lambda is responsible for the insertion of viral DNA into the confined space within the capsid. The enzyme is composed of the virally encoded proteins gpA (73.3 kDa) and gpNu1 (20.4 kDa) isolated as a gpA(1).gpNu1(2) holoenzyme complex. Lambda terminase possesses a site-specific nuclease activity, an ATP-dependent DNA strand-separation activity, and an ATPase activity that must work in concert to effect genome packaging. We have previously characterized the ATPase activity of the holoenzyme and have identified catalytic active sites in each enzyme subunit [Tomka and Catalano (1993) Biochemistry 32, 11992-11997; Hwang et al. (1996) Biochemistry 35, 2796-2803]. We have noted that GTP stimulates the ATPase activity of the enzyme, and terminase-mediated GTP hydrolysis has been observed. The studies presented here describe a kinetic analysis of the GTPase activity of lambda terminase. GTP hydrolysis by the enzyme requires divalent metal, is optimal at alkaline pH, and is strongly inhibited by salt. Interestingly, while GTP can bind to the enzyme in the absence of DNA, GTP hydrolysis is strictly dependent on the presence of polynucleotide. Unlike ATP hydrolysis that occurs at both subunits of the holoenzyme, a single catalytic site is observed in the steady-state kinetic analysis of GTPase activity (k(cat) approximately 37 min(-)(1); K(m) approximately 500 microM). Moreover, while GTP stimulates ATP hydrolysis (apparent K(D) approximately 135 microM for GTP binding), all of the adenosine nucleotides examined strongly inhibit the GTPase activity of the enzyme. The data presented here suggest that the two "NTPase" catalytic sites in terminase holoenzyme communicate, and we propose a model describing allosteric interactions between the two sites. The biological significance of this interaction with respect to the assembly and disassembly of the multiple nucleoprotein packaging complexes required for virus assembly is discussed.     

Molecular basis for G protein control of the prokaryotic ATP sulfurylase

Sulfate assimilation is a critical component of both primary and secondary metabolism. An essential step in this pathway is the activation of sulfate through adenylation by the enzyme ATP sulfurylase (ATPS), forming adenosine 5'-phosphosulfate (APS). Proteobacterial ATPS overcomes this energetically unfavorable reaction by associating with a regulatory G protein, coupling the energy of GTP hydrolysis to APS formation. To discover the molecular basis of this unusual role for a G protein, we biochemically characterized and solved the X-ray crystal structure of a complex between Pseudomonas syringae ATPS (CysD) and its associated regulatory G protein (CysN). The structure of CysN*D shows the two proteins in tight association; however, the nucleotides bound to each subunit are spatially segregated. We provide evidence that conserved switch motifs in the G domain of CysN allosterically mediate interactions between the nucleotide binding sites. This structure suggests a molecular mechanism by which conserved G domain architecture is used to energetically link GTP turnover to the production of an essential metabolite.     

Thr-431 and Arg-433 are part of a conserved sequence motif of the glutamine amidotransferase domain of CTP synthases and are involved in GTP activation of the Lactococcus lactis enzyme

A conserved sequence motif within the class 1 glutamine amidotransferase (GATase) domain of CTP synthases was identified. The sequence motif in the Lactococcus lactis enzyme is (429)GGTLRLG(435). This motif was present only in CTP synthases and not in other enzymes that harbor the GATase domain. Therefore, it was speculated that this sequence was involved in GTP activation of CTP synthase. Other members of the GATase protein family are not activated allosterically by GTP. Residues Thr-431 and Arg-433 were changed by site directed mutagenesis to the sterically similar residues valine and methionine, respectively. The resulting enzymes, T431V and R433M, had both lost the ability for GTP to activate the uncoupled glutaminase activity and showed reduced GTP activation of the glutamine-dependent CTP synthesis reaction. The T431V enzyme had a similar activation constant, K(A), for GTP, but the activation was only 2-3-fold compared with 35-fold for the wild type enzyme. The R433M enzyme was found to have a 10-15-fold lower K(A) for GTP and a concomitant decrease in V(app). The activation by GTP of this enzyme was about 7-fold. The kinetic parameters for saturation with ATP, UTP, and NH(4)Cl were similar for wild type and mutant enzymes, except that the R433M enzyme only had half the V(app) of the wild type enzyme when NH(4)Cl was the amino donor. The mutant enzymes T431V and R433M apparently had not lost the ability to bind GTP, but the signal transmitted through the enzyme to the active sites upon binding of the allosteric effector was clearly disrupted in the mutant enzymes.     

A novel reaction catalyzed by unadenylylated glutamine synthetase from Escherichia coli. AMP-dependent synthesis of pyrophosphate and L-Glutamate from orthophosphate and L-glutamine

The unadenylylated, manganese form of glutamine synthetase (L-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2 from Escherichia coli catalyzes a novel, AMP-dependent (reversible) synthesis of pyrophosphate and L-glutamate from orthophosphate and L-glutamine: Formula (See Text). The hydrolysis of the L-glutamine amide bond is coupled to the stoichiometric synthesis of pyrophosphate, although as PPi accumulates, additional hydrolysis of L-glutamine occurs in a secondary reaction catalyzed by the [manganese x enzyme x AMP x PPi] complex. The synthesis of PPi probably occurs at the subunit catalytic site in the positions normally occupied by the beta, gamma-phosphates of ATP. To promote PPi synthesis, AMP apparently binds to the subunit catalytic site rather than to the allosteric inhibitor site; equilibrium binding results suggest that Pi directs the binding of AMP to the active site. In this reaction, Mg2+ will not substitute for Mn2+, and adenylylated glutamine synthetase is inactive. Pyrophosphate is synthesized by the unadenylylated, manganese enzyme at approximately 2% of the rate of that of ATP in the reverse biosynthetic reaction. If P1 is replaced by arsenate, the enzymatic rate of the AMP-supported hydrolysis of L-glutamine is 100-fold faster than is PPi synthesis and is one-half the rate of the ADP-supported, irreversible arsenolysis of L-glutamine. This latter activity also is supported by GMP and IMP, suggesting that the catalytic site of glutamine synthetase has a rather broad specificity for the nucleotide base. The reactions supported by AMP directly relate to the mechanism of glutamine synthetase catalysis.     

A mutational analysis of the PD...D/EXK motif suggests that McrC harbors the catalytic center for DNA cleavage by the GTP-dependent restriction enzyme McrBC from Escherichia coli

McrBC is a unique restriction enzyme which binds specifically to the bipartite recognition sequence R(m)CN( approximately )(30)(-)( approximately )(2000)R(m)C and in the presence of GTP translocates the DNA and cleaves both strands at multiple positions within the two R(m)C "half-sites". It is known that McrBC is composed of two subunits: McrB which binds and hydrolyzes GTP and specifically interacts with DNA and McrC whose function is not clear but which has been suspected to harbor the catalytic center for DNA cleavage. A multiple-sequence alignment of the amino acid sequence of Escherichia coli McrC and of six presumably homologous open reading frames from various bacterial species shows that a sequence motif found in many restriction enzymes, but also in other nucleases, the PD.D/EXK motif, is conserved among these sequences. A mutational analysis, in which the carboxylates (aspartic acid in McrC) of this motif were substituted with alanine or asparagine and lysine was substituted with alanine or arginine, strongly suggests that Asp244, Asp257, and Lys259 represent the catalytic center of E. coli McrC. Whereas the variants D244A (or -N), D257A (or -N), and K259A are inactive in DNA cleavage (K259R has residual DNA cleavage activity), they interact with McrB like wild-type McrC, as can be deduced from the finding that they stimulate the McrB-catalyzed GTP hydrolysis to the same extent as wild-type McrC. Thus, whereas McrC variants defective in DNA cleavage can stimulate the GTPase activity of McrB, the DNase activity of McrC is not supported by McrB variants defective in GTP hydrolysis.     

PPi-dependent phosphofructotransferase (phosphofructokinase) activity in the mollicutes (mycoplasma) Acholeplasma laidlawii.

A PPi-dependent phosphofructotransferase (PPi-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.90) which catalyzes the conversion of fructose 6 phosphate (F-6-P) to fructose 1,6-bisphosphate (F-1, 6-P2) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (430-fold). PPi was required as the phosphate donor. ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, dUTP, ITP, TTP, ADP, or Pi could not substitute for PPi. The PPi-dependent reaction (2.0 mM PPi) was not altered in the presence of any of these nucleotides (2.0 mM) or in the presence of smaller (less than or equal to 300 microM) amounts of fructose 2,6-bisphosphate, (NH4)2SO4, AMP, citrate, GDP, or phosphoenolpyruvate. Mg2+ and a pH of 7.4 were required for maximum activity. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 74,000 and a sedimentation coefficient of 6.7. A second form of the enzyme (molecular weight, 37,000) was detected, although in relatively smaller amounts, by using Blue Sepharose matrix when performing electrophoresis experiments. The back reaction, F-1, 6-P2 to F-6-P, required Pi; arsenate could substitute for Pi, but not PPi or any other nucleotide tested. The computer-derived kinetic constants (+/- standard deviation) for the reaction in the PPi-driven direction of F-1, 6-P2 were as follows: v, 38.9 +/- 0.48 mM min-1; Ka(PPi), 0.11 +/- 0.04 mM; Kb(F-6-P), 0.65 +/- 0.15 mM; and Kia(PPi), 0.39 +/- 0.11 mM. A. laidlawii B-PG9 required PPi not only for the PPi-phosphofructotransferase reaction which we describe but also for purine nucleoside kinase activity. a dependency unknown in any other organism. In A. laidlawii B-PG9, the PPi requirement may be met by reactions in this organism already known to synthesize PPi (e.g., dUTPase and purine nucleobase phosphoribosyltransferases). In almost all other cells, the conversion of F-6-P to F-1,6-P2 is ATP dependent, and the reaction is generally considered to be the rate-limiting step of glycolysis. The ability of A. laidlawii B-PG9 and one other acholeplasma to use PPi instead of ATP as an energy source may offer these cytochrome-deficient organisms some metabolic advantage and may represent a conserved metabolic remnant of an earlier evolutionary process.     

Evidence for the participation of independent translocation for phosphate and glucose 6-phosphate in the microsomal glucose-6-phosphatase system. Interactions of the system with orthophosphate, inorganic pyrophosphate, and carbamyl phosphate

The interactions of Pi, PPi, and carbamyl-P with the hepatic glucose-6-phosphatase system were studied in intact and detergent-disrupted microsomes. Penetration of PPi and carbamyl-P into intact microsomes was evidenced by their reactions with the enzyme located exclusively on the luminal surface. Lack of effects of carbonyl cyanide m-chlorophenylhydrazone and valinomycin + KCl indicated that pH gradients and/or membrane potentials that could influence the kinetics of the system are not generated during metabolism of PPi and glucose-6-P by intact microsomes. With disrupted microsomes, only competitive interactions were seen among glucose-6-P, Pi, PPi, and carbamyl-P. With intact microsomes, Pi, PPi, and carbamyl-P were relatively weak, noncompetitive inhibitors of glucose-6-phosphatase, and PPi hydrolysis was inhibited competitively by Pi and carbamyl-P but noncompetitively by glucose-6-P. Analysis of the kinetic data in combination with findings from other studies that a variety of inhibitors of the glucose-6-P translocase (T1) does not affect PPi hydrolysis provide compelling evidence that permeability of microsomes to Pi, PPi, and carbamyl-P is mediated by a second translocase (T2). Some properties of the microsomal anion transporters are described. If the characteristics of the glucose-6-phosphatase system as presently defined in intact microsomes apply in vivo, glucose-6-P hydrolysis appears to be the predominant, if not the exclusive, physiologic function of the system. Both the "noncompetitive character" and the relative ineffectiveness of Pi as an inhibitor of glucose-6-phosphatase of intact microsomes result from the rate limitation imposed by T1 that prevents equilibration of glucose-6-P across the membrane. In microsomes from fed rats, where T1 is less rate restricting, about one-half as much Pi was required to give 50% inhibition compared with microsomes from fasted or diabetic rats. Thus, any treatment or agent that alters the kinetic relationship between transport and hydrolysis of glucose-6-P (e.g. endocrine or nutritional status) is an essential consideration in analyses of kinetic data for the glucose-6-phosphatase system.     

Stabilization of the enzyme--substrate complex of the mutant Asp-67Asn inorganic pyrophosphatase from Escherichia coli by fluoride ions

Magnesium-supported PPi hydrolysis by the mutant Asp-67Asn E. coli pyrophosphatase at saturating PPi and metal-activator concentrations in the presence of NaF is followed by a gradual decrease in the initial rate of PPi hydrolysis. The reaction occurs in two steps: first a complex containing enzyme, pyrophosphate, magnesium, and fluoride ions is immediately formed, then its conformation changes slowly. This enzyme--substrate complex stabilized by fluoride is partially active and can be isolated by the removal of excess fluoride by gel-filtration.     

Biosynthesis of pteridines. Reaction mechanism of GTP cyclohydrolase I

GTP cyclohydrolase I catalyses the hydrolytic release of formate from GTP followed by cyclization to dihydroneopterin triphosphate. The enzymes from bacteria and animals are homodecamers containing one zinc ion per subunit. Replacement of Cys110, Cys181, His112 or His113 of the enzyme from Escherichia coli by serine affords catalytically inactive mutant proteins with reduced capacity to bind zinc. These mutant proteins are unable to convert GTP or the committed reaction intermediate, 2-amino-5-formylamino-6-(beta-ribosylamino)-4(3H)-pyrimidinone 5'-triphosphate, to dihydroneopterin triphosphate. The crystal structures of GTP complexes of the His113Ser, His112Ser and Cys181Ser mutant proteins determined at resolutions of 2.5A, 2.8A and 3.2A, respectively, revealed the conformation of substrate GTP in the active site cavity. The carboxylic group of the highly conserved residue Glu152 anchors the substrate GTP, by hydrogen bonding to N-3 and to the position 2 amino group. Several basic amino acid residues interact with the triphosphate moiety of the substrate. The structure of the His112Ser mutant in complex with an undefined mixture of nucleotides determined at a resolution of 2.1A afforded additional details of the peptide folding. Comparison between the wild-type and mutant enzyme structures indicates that the catalytically active zinc ion is directly coordinated to Cys110, Cys181 and His113. Moreover, the zinc ion is complexed to a water molecule, which is in close hydrogen bond contact to His112. In close analogy to zinc proteases, the zinc-coordinated water molecule is suggested to attack C-8 of the substrate affording a zinc-bound 8R hydrate of GTP. Opening of the hydrated imidazole ring affords a formamide derivative, which remains coordinated to zinc. The subsequent hydrolysis of the formamide motif has an absolute requirement for zinc ion catalysis. The hydrolysis of the formamide bond shows close mechanistic similarity with peptide hydrolysis by zinc proteases.     

Thermodynamics, kinetics, and mechanism in yeast inorganic pyrophosphatase catalysis of inorganic pyrophosphate: inorganic phosphate equilibration

We have developed two methods for quantitatively measuring inorganic pyrophosphate (PPi) in the presence of 10(3)--10(4) molar excesses of inorganic phosphate (Pi) and used them to measure the extent of enzyme-bound pyrophosphate (EPPi) formation in solutions of yeast inorganic pyrophosphatase and Pi. We have also measured the rate of enzyme-catalyzed H2O--phosphate oxygen exchange. We find both processes to have essentially identical dependence on Mg2+ and Pi concentrations, thus providing important confirmation for the recent proposal by Janson et al. (1979) that oxygen exchange proceeds via EPPi formation. Our results are consistent with a model in which three Mg2+ per active site are required for EPPi formation but inconsistent with a model requiring only two Mg2+ per active site and permit the formulation of an overall scheme for inorganic pyrophosphatase catalysis of PPi--Pi equilibration as well as the evaluation of equilibrium and rate constants in this scheme. The major results and conclusions of our work are the following: (a) the equilibrium constant for PPi (enzyme-bound) in equilibrium with 2Pi (enzyme-bound) is 4.8; (b) following PPi hydrolysis, the first released Pi contains an oxygen from solvent water; (c) the steps for PPi hydrolysis on the enzyme and for release of both product Pi's are all partially rate determining in overall enzyme-catalyzed PPi hydrolysis; (d) PPi formation on the enzyme is rate determining for H2O--Pi oxygen exchange; (e) PPi dissociation from the enzyme is very slow and is the rate-determining step in Pi--PPi exchange (Cohn, 1958; Janson et al., 1979). This also accounts for the observation that the calculated dissociation constant for MgPPi complex binding to enzyme is considerably lower than the measured Km for enzyme-catalyzed MgPPi hydrolysis.     

RNA capping by HeLa cell RNA guanylyltransferase. Characterization of a covalent protein-guanylate intermediate

RNA capping by partially purified HeLa cell GTP:RNA guanylyltransferase has been shown to occur in the following sequence of two partial reactions involving a covalent protein-guanylate intermediate: (i) E(P68) + GTP in equilibrium E(P68-GMP) + PPi (ii) E(P68-GMP) + ppRNA in equilibrium GpppRNA + E(P68) Initially, the enzyme reacts with GTP in the absence of an RNA cap acceptor to form a covalent protein-guanylate complex. This complex consists of a GMP residue linked via a phosphoamide bond to a Mr = 68,000 protein. The enzyme then transfers the guanylate residue from the Mr = 68,000 polypeptide to the 5' end of diphosphate-terminated poly(a) to yield the capped derivative GpppA(pA)n. Both partial reactions have been shown to be reversible. In the reverse of Reaction i, E(P68--GMP) reacts with PPi to regenerate GTP. In the reverse of Reaction ii, the enzyme catalyzes the transfer of the 5'-GMP from capped RNA to the Mr = 68,000 protein to form protein-guanylate complex. A divalent cation is required for both partial reactions. The Mr = 68,000 protein is presumed to be a subunit of the HeLa guanylyltransferase. This interpretation is consistent with the sedimentation coefficient of 4.2 S of the native enzyme. Preliminary studies of RNA guanylyltransferase from mouse myeloma tumors suggest a similar mechanism of transguanylylation involving a Mr = 68,000 protein-guanylate complex. These data, in conjunction with previous studies of vaccinia virus guanylyltransferase (Shuman, S., and Hurwitz, J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 187-191) suggests that covalent GMP-enzyme intermediates may be a general feature of the RNA capping reaction.     

Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis

The gene thiI encodes a protein (ThiI) that plays a role in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine, but the reaction catalyzed by ThiI remains undetermined. Based upon sequence alignments, ThiI shares a unique "P-loop" motif with the PPi synthetase family, four enzymes that catalyze adenylation and subsequent substitution of carbonyl oxygens. To test whether or not this motif is critical for ThiI function, the Asp in the motif was converted to Ala (D189A), and a screen for in vivo 4-thiouridine production revealed the altered enzyme to be inactive. Further scrutiny of sequence data and the crystal structures of two members of the PPi synthetase family implicated Lys321 in the proposed adenylation function of ThiI, and the critical nature of Lys321 has been demonstrated by site-directed mutagenesis and genetic screening. Our results, then, indicate that ThiI catalyzes the adenylation of a substrate at the expense of ATP, a narrowing of possible reactions that provides a strong new basis for deducing the early steps in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine.     

Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) complexed with GMP: evidence for a substrate-induced transferase active site.

The X-ray crystal structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) from Salmonella typhimurium bound to GMP has been determined by molecular replacement to 2.2 A resolution. CobU is a bifunctional enzyme, which catalyzes the phosphorylation of the 1-amino-O-2-propanol side chain of the adenosylcobinamide ring and subsequently functions as a guanylyltransferase to form adenosylcobinamide.GDP. The transferase activity involves a covalent enzyme-guanylyl intermediate that is most likely a phosphoramidate linkage to His(46). Previous studies have shown that the enzyme is a homotrimer and adopts a pinwheel shape. Each subunit consists of a single domain of six parallel beta-strands and one antiparallel strand flanked on either side by a total of five alpha-helices and one helical turn. Interestingly, His(46) in the apoenzyme is located a considerable distance from the kinase active site or P-loop motif and is solvent-exposed [Thompson, T. B., et al. (1998) Biochemistry 37, 7686-7695]. To examine the structural relationship of the two active sites, CobU was cocrystallized with GTP and pyrophosphate. Crystals belong to space group P2(1)2(1)2(1) with the following unit cell dimensions: a = 58. 4 A, b = 87.8 A, and c = 101.6 A. The structure shows electron density for the hydrolysis product GMP rather than the expected covalent guanylyl intermediate which appears to have been hydrolyzed in the crystal lattice. Even so, CobU exhibits a substantial conformational rearrangement. The helix axis containing His(46), the site of guanylylation, rotates 30 degrees and translates 11 A relative to the apo structure and is accompanied by compensatory unwinding and rewinding at the helix ends to allow the induction of a guanosine binding pocket between beta-strand 2 and alpha-helix 2. This conformational change brings the C(alpha) of His(46) approximately 10 A closer to the P-loop motif such that a phosphate ion located in the P-loop is only 6 A from the alpha-phosphate of GMP. This suggests that the P-loop motif may be used to coordinate the terminal phosphates in both the transferase and kinase reactions and implies that the active sites for both reactions overlap.     

Role of Individual Domains and Identification of Internal Gap in Human Guanylate Binding Protein-1

Unlike other GTPases, interferon-gamma-induced human guanylate binding protein-1 has the ability to hydrolyze GTP to both GDP and GMP, with GMP being the major product of the reaction. This protein has two domains, an N-terminal globular domain and a C-terminal helical domain. These two domains are connected by a short intermediate region consisting of a two-stranded β-sheet and a helix. As human guanylate binding protein-1 has been shown to undergo stimulated GTPase activity without external GTPase-activating protein, we sought to understand the roles of each of the two individual domains, the intermediate region, a conserved motif (¹⁰³DXEKGD¹⁰⁸), and the mechanism of the stimulation of GTPase activity. The steady-state assays using radiolabeled [α-³²P]GTP on the wild-type protein suggest that the stimulation of activity primarily occurs during the cleavage of the second phosphate of GTP rather than the first, through allosteric interaction. Using several truncated and mutant proteins, we demonstrate for the first time that both the α-helix of the intermediate region and the ¹⁰³DXEKGD¹⁰⁸ motif play critical roles for the hydrolysis to GMP, but they appear to act in different ways: α-helix acts through structural stabilization by allosteric interaction and, thus, acts as an internal GTPase-activating protein, whereas the motif might act by providing necessary catalytic residues. Our data also show that the N-terminal globular domain is able to perform only the first catalysis (GTP to GDP, an activity associated with basal level), but the helical domain in the full-length protein stimulates the hydrolysis of GTP to GMP with higher GMP formation by preventing the dissociation of GDP-bound enzyme dimer.     

Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis. The role of the allosteric activator GTP incoupling between glutamine hydrolysis and CTP synthesis.

CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself.     

Anatomy of an energy-coupling mechanism--the interlocking catalytic cycles of the ATP sulfurylase-GTPase system

ATP sulfurylase, from Escherichia coli K-12, conformationally couples the rates and chemical potentials of the two reactions that it catalyzes, GTP hydrolysis and activated sulfate synthesis. The enzyme is rare among such coupling systems in that it links the potentials of small-molecule chemistries to one another, rather than to vectorial motion. The pre-steady-state stages of the catalytic cycle of ATP sulfurylase were studied using tools capable of distinguishing between enzyme-bound and solution-phase product for each of the four products of the enzyme. The study reveals that the two chemistries are linked at multiple points in the reaction coordinate. Linking begins with an isomerization prior to chemistry that initiates an ordered cleavage of the beta,gamma and alpha,beta bonds of GTP and ATP, respectively; the rates of these three sequential events increase successively, causing them to appear simultaneous. Linking is again seen in the late stages of the catalytic cycle: product release is ordered with P(i) departing prior to either GDP or PP(i). Release rate constants are determined for each product and used to construct a quantitative model of the mechanism that accurately predicts the behavior of this complex system.     

The structure of ADP-ribose pyrophosphatase reveals the structural basis for the versatility of the Nudix family

Regulation of cellular levels of ADP-ribose is important in preventing nonenzymatic ADP-ribosylation of proteins. The Escherichia coli ADP-ribose pyrophosphatase, a Nudix enzyme, catalyzes the hydrolysis of ADP-ribose to ribose-5-P and AMP, compounds that can be recycled as part of nucleotide metabolism. The structures of the apo enzyme, the active enzyme and the complex with ADP-ribose were determined to 1.9 A, 2.7 A and 2.3 A, respectively. The structures reveal a symmetric homodimer with two equivalent catalytic sites, each formed by residues of both monomers, requiring dimerization through domain swapping for substrate recognition and catalytic activity. The structures also suggest a role for the residues conserved in each Nudix subfamily. The Nudix motif residues, folded as a loop-helix-loop tailored for pyrophosphate hydrolysis, compose the catalytic center; residues conferring substrate specificity occur in regions of the sequence removed from the Nudix motif. This segregation of catalytic and recognition roles provides versatility to the Nudix family.     

Substrate, metal and template effects on inhibition of bacteriophage-qbeta ribonucleic acid polymerase by ortho- and pyro-phosphate.

Two reactions of bacteriophage-Qbeta RNA polymerase with synthetic templates were characterized and used to study the effects of substrate, metal and template on inhibition by Pi and PPi. Analysis of the poly(C)-dependent reaction yielded results on kinetics, GTP-dependence, preference for Mn2+ over Mg2+, and Michaelis constants for template similar to those in the literature. New data are provided for the poly(U2,C)-dependent reaction. Our results suggest that GTP and Mn2+ can form relatively stable complexes with the polymerase and that such complexes change the interaction of the enzyme with the inhibitors, Pi and PPi.