Limited proteolysis of Escherichia coli cytidine 5'-triphosphate synthase. Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis.

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH3-dependent CTP formation. For these mutants, the values of kcat/Km and kcat for glutamine-dependent CTP formation were reduced approximately 20-fold and approximately 10-fold, respectively, relative to wild-type enzyme; however, the value of Km for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold. This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding.     

Competition between ammonia derived from internal glutamine hydrolysis and hydroxylamine present in the solution for incorporation into UTP as catalysed by Lactococcus lactis CTP synthase

CTP synthase catalyses the reaction: glutamine+UTP+ATP --> glutamate+CTP+ADP+P(i). The reaction is greatly stimulated by the allosteric binding of GTP. In addition to glutamine that is hydrolysed by the enzyme to ammonia and glutamate, CTP synthase will also utilise external sources of amino donors such as NH(4)Cl. This reaction is no longer dependent on allosteric activation by GTP. Hydroxylamine is also a substrate for Lactococcus lactis CTP synthase and results in the formation of N4-OH CTP. This product has the feature that it absorbs at 300nm where CTP absorption was shown to be greatly reduced and enabled the determination of N4-OH CTP formation in the presence of CTP synthesis derived from glutamine hydrolysis. Differences in initial rates determined for the hydroxylamine dependent reaction at 291nm in the presence and absence of glutamine and GTP were ascribed to simultaneous CTP and N4-OH CTP synthesis in the presence of these compounds. A characterisation of the apparent inhibition by GTP and glutamine of N4-OH CTP synthesis determined at 300nm showed that glutamine dependent CTP synthesis occurs at a rate of about 60% of that in the absence of hydroxylamine. GTP dependent inhibition of the ammonium chloride dependent reaction of L. lactis CTP synthase by the glutamine analog glutamate gamma-semialdehyde showed a partial inhibition with a maximum inhibition of about 60%. These results are interpreted in terms of a "half of the sites" mechanism for glutamine hydrolysis on CTP synthase.     

Alternative substrates for wild-type and L109A E. coli CTP synthases: kinetic evidence for a constricted ammonia tunnel.

Cytidine 5'-triphosphate (CTP) synthase catalyses the ATP-dependent formation of CTP from uridine 5'-triphosphate using either NH(3) or l-glutamine as the nitrogen source. The hydrolysis of glutamine is catalysed in the C-terminal glutamine amide transfer domain and the nascent NH(3) that is generated is transferred via an NH(3) tunnel [Endrizzi, J.A., Kim, H., Anderson, P.M. & Baldwin, E.P. (2004) Biochemistry43, 6447-6463] to the active site of the N-terminal synthase domain where the amination reaction occurs. Replacement of Leu109 by alanine in Escherichia coli CTP synthase causes an uncoupling of glutamine hydrolysis and glutamine-dependent CTP formation [Iyengar, A. & Bearne, S.L. (2003) Biochem. J.369, 497-507]. To test our hypothesis that L109A CTP synthase has a constricted or a leaky NH(3) tunnel, we examined the ability of wild-type and L109A CTP synthases to utilize NH(3), NH(2)OH, and NH(2)NH(2) as exogenous substrates, and as nascent substrates generated via the hydrolysis of glutamine, gamma-glutamyl hydroxamate, and gamma-glutamyl hydrazide, respectively. We show that the uncoupling of the hydrolysis of gamma-glutamyl hydroxamate and nascent NH(2)OH production from N(4)-hydroxy-CTP formation is more pronounced with the L109A enzyme, relative to the wild-type CTP synthase. These results suggest that the NH(3) tunnel of L109A, in the presence of bound allosteric effector guanosine 5'-triphosphate, is not leaky but contains a constriction that discriminates between NH(3) and NH(2)OH on the basis of size.     

Lid L11 of the glutamine amidotransferase domain of CTP synthase mediates allosteric GTP activation of glutaminase activity

GTP is an allosteric activator of CTP synthase and acts to increase the k(cat) for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) domain of CTP synthase. In the GATase domain of the recently published structures of the Escherichia coli and Thermus thermophilus CTP synthases a loop region immediately proceeding amino acid residues forming the oxy-anion hole and named lid L11 is shown for the latter enzyme to be flexible and change position depending on the presence or absence of glutamine in the glutamine binding site. Displacement or rearrangement of this loop may provide a means for the suggested role of allosteric activation by GTP to optimize the oxy-anion hole for glutamine hydrolysis. Arg359, Gly360 and Glu362 of the Lactococcus lactis enzyme are highly conserved residues in lid L11 and we have analyzed their possible role in GTP activation. Characterization of the mutant enzymes R359M, R359P, G360A and G360P indicated that both Arg359 and Gly360 are involved in the allosteric response to GTP binding whereas the E362Q enzyme behaved like wild-type enzyme. Apart from the G360A enzyme, the results from kinetic analysis of the enzymes altered at position 359 and 360 showed a 10- to 50-fold decrease in GTP activation of glutamine dependent CTP synthesis and concomitant four- to 10-fold increases in K(A) for GTP. The R359M, R359P and G360P also showed no GTP activation of the uncoupled glutaminase reaction whereas the G360A enzyme was about twofold more active than wild-type enzyme. The elevated K(A) for GTP and reduced GTP activation of CTP synthesis of the mutant enzymes are in agreement with a predicted interaction of bound GTP with lid L11 and indicate that the GTP activation of glutamine dependent CTP synthesis may be explained by structural rearrangements around the oxy-anion hole of the GATase domain.     

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.     

Investigation of the mechanism of CTP synthetase using rapid quench and isotope partitioning methods

The UTP-dependent ATPase reaction and the glutamine-dependent overall reaction of Escherichia coli CTP synthetase have been studied by rapid quench and isotope partitioning kinetics. The effect of GTP, an allosteric effector, on the pre-steady-state kinetics of both reactions has also been examined. The time courses of the UTP-dependent ATPase reaction in the presence and absence of GTP are both characterized by a burst of acid-labile phosphate equivalent to 0.93 and 0.43 subunits, respectively. The time course of the glutamine-dependent reaction in the absence of GTP is also characterized by a burst of acid-labile phosphate corresponding to 0.8 subunit; however, in the presence of GTP, no burst was observed. These results along with positional isotope exchange experiments [von der Saal, W., Anderson, P. M., & Villafranca, J. J. (1985) J. Biol. Chem. 260, 14997] provide evidence that the mechanism of CTP formation involves phosphorylation of UTP followed by attack of NH3, and finally release of phosphate, producing CTP, ADP, and Pi. A kinetic model for the first stages of the enzymatic reaction was developed from the rapid quench data, and the internal equilibrium constant for the formation of the phosphorylated UTP intermediate was determined. The internal equilibrium constants for the UTP-dependent reaction in the presence and absence of GTP were found to be 1.1 and 18, respectively. By contrast, the internal equilibrium constant for the reaction in the presence of glutamine was 50. Thus, the presence of glutamine shifts the internal equilibrium constant to favor formation of the phosphorylated UTP intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural requirements for the activation of Escherichia coli CTP synthase by the allosteric effector GTP are stringent, but requirements for inhibition are lax

Cytidine 5'-triphosphate synthase catalyzes the ATP-dependent formation of CTP from UTP using either NH(3) or l-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Gln-dependent CTP formation at concentrations of >0.15 mM and NH(3)-dependent CTP formation at all concentrations. A structure-activity study using a variety of GTP and guanosine analogues revealed that only a few GTP analogues were capable of activating Gln-dependent CTP formation to varying degrees: GTP approximately 6-thio-GTP > ITP approximately guanosine 5'-tetraphosphate > O(6)-methyl-GTP > 2'-deoxy-GTP. No activation was observed with guanosine, GMP, GDP, 2',3'-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5'-triphosphate, 2'-OH, and 3'-OH are required for full activation. The 2-NH(2) group plays an important role in binding recognition, whereas substituents at the 6-position play an important role in activation. The presence of a 6-NH(2) group obviates activation, consistent with the inability of ATP to substitute for GTP. Nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibited NH(3)- and Gln-dependent CTP formation (often in a cooperative manner) to a similar extent (IC(50) approximately 0.2-0.5 mM). This inhibition appeared to be due solely to the purine base and was relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC(50) approximately 4-12 mM). 8-Oxoguanosine was the best inhibitor identified (IC(50) = 80 microM). Our findings suggest that modifying 2-aminopurine or 2-aminopurine riboside may serve as an effective strategy for developing cytidine 5'-triphosphate synthase inhibitors.     

Activation and inhibition of CTP synthase from Trypanosoma brucei, the causative agent of African sleeping sickness

CTP Synthase from Trypanosoma brucei (TbCTPS) catalyzes the conversion of UTP to CTP and is a recognized target for the development of antiprotozoal agents. GTP activates glutamine-dependent CTP formation catalyzed by TbCTPS at concentrations below 0.2 mM, but inhibits this activity at concentrations above 0.2 mM. TbCTPS catalyzes ammonia-dependent CTP formation, which is inhibited by purine derivatives such as GTP, guanosine, caffeine, and uric acid with IC(50) values of 460, 380, 480, and 100 μM, respectively. These observations suggest that the purine ring may serve as a useful scaffold for the development of inhibitors of trypanosomal CTP synthase.     

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.     

“Pinching” the ammonia tunnel of CTP synthase unveils coordinated catalytic and allosteric-dependent control of ammonia passage

Molecular gates within enzymes often play important roles in synchronizing catalytic events. We explored the role of a gate in cytidine-5′-triphosphate synthase (CTPS) from Escherichia coli. This glutamine amidotransferase catalyzes the biosynthesis of CTP from UTP using either l-glutamine or exogenous NH₃ as a substrate. Glutamine is hydrolyzed in the glutaminase domain, with GTP acting as a positive allosteric effector, and the nascent NH₃ passes through a gate located at the end of a ~25-Å tunnel before entering the synthase domain where CTP is generated. Substitution of the gate residue Val 60 by Ala, Cys, Asp, Trp, or Phe using site-directed mutagenesis and subsequent kinetic analyses revealed that V60-substitution impacts glutaminase activity, nucleotide binding, salt-dependent inhibition, and inter-domain NH₃ transport. Surprisingly, the increase in steric bulk present in V60F perturbed the local structure consistent with “pinching” the tunnel, thereby revealing processes that synchronize the transfer of NH₃ from the glutaminase domain to the synthase domain. V60F had a slightly reduced coupling efficiency at maximal glutaminase activity that was ameliorated by slowing down the glutamine hydrolysis reaction, consistent with a “bottleneck” effect. The inability of V60F to use exogenous NH₃ was overcome in the presence of GTP, and more so if CTPS was covalently modified by 6-diazo-5-oxo-l-norleucine. Use of NH₂OH by V60F as an alternative bulkier substrate occurred most efficiently when it was concomitant with the glutaminase reaction. Thus, the glutaminase activity and GTP-dependent activation act in concert to open the NH₃ gate of CTPS to mediate inter-domain NH₃ transport.     

Substrate inhibition of Lactococcus lactis cytidine 5'-triphosphate synthase by ammonium chloride is enhanced by salt-dependent tetramer dissociation

Cytidine 5(')-triphosphate (CTP) synthase (EC 6.4.3.2) catalyzes the transfer of an amino group to the 4 position of uridine 5(')-triphosphate (UTP) to yield CTP. The reaction proceeds by activation of the base moiety of UTP by adenosine 5(')-triphosphate (ATP)-dependent phosphorylation. The activated intermediate reacts with NH(3) in the solution or is obtained by hydrolysis of glutamine. The Lactococcus lactis CTP synthase shows significant differences from the enzymes from Escherichia coli, yeast, and mammals. One is the apparent stability of the L. lactis CTP synthase tetramer in the absence of the nucleotides ATP and UTP. This condition causes the E. coli, yeast, and mammal enzymes to dissociate into dimers. However, the L. lactis CTP synthase shows substrate inhibition by NH(4)Cl that coincides with the range of NH(4)Cl concentrations that apparently dissociates tetrameric enzyme into dimers. Even though regular substrate inhibition was observed with NH(4)Cl when the ionic strength was held constant, a significant part of the inhibition could be shown to be due to the increase in ionic strength with increasing substrate concentration. Since the substrate inhibition by NH(4)Cl was relieved by increasing the equimolar ATP and UTP concentrations, it appeared that the substrate nucleotides stabilized the tetramer in a manner similar to that found in the absence of salt for other CTP synthases. In contrast to the suggested hydrophobic nature of the tetramer interactions in E. coli CTP synthase, the dissociation of the L. lactis CTP synthase tetramer in response to an increase in ionic strength suggests that the tetramer is stabilized by ionic interactions.     

Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets

Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics.     

The role of lysine residues 297 and 306 in nucleoside triphosphate regulation of E. coli CTP synthase: inactivation by 2',3'-dialdehyde ATP and mutational analyses

Cytidine 5'-triphosphate synthase (CTPS) catalyzes the ATP-dependent formation of CTP from UTP using either NH3 or L-glutamine as the source of nitrogen. To identify the location of the ATP-binding site within the primary structure of E. coli CTPS, we used the affinity label 2',3'-dialdehyde adenosine 5'-triphosphate (oATP). oATP irreversibly inactivated CTPS in a first-order, time-dependent manner while ATP protected the enzyme from inactivation. In the presence of 10 mM UTP, the values of k(inact) and K(I) were 0.054 +/- 0.001 min(-1) and 3.36 +/- 0.02 mM, respectively. CTPS was labeled using (2,8-3H)oATP and subsequently subjected to trypsin-catalyzed proteolysis. The tryptic peptides were separated using reversed-phase HPLC, and two peptides were identified using N-terminal sequencing (S(492)GDDQLVEIIEVPNH(506) and Y(298)IELPDAY(K(306)) in a 5:1 ratio). The latter suggested that Lys 306 had been modified by oATP. Replacement of Lys 306 by alanine reduced the rate of oATP-dependent inactivation (k(inact) = 0.0058 +/- 0.0005 min(-1), K(I) = 3.7 +/- 1.3 mM) and reduced the apparent affinity of CTPS for both ATP and UTP by approximately 2-fold. The efficiency of K306A-catalyzed glutamine-dependent CTP formation was also reduced 2-fold while near wild-type activity was observed when NH3 was the substrate. These findings suggest that Lys 306 is not essential for ATP binding, but does play a role in bringing about the conformational changes that mediate interactions between the ATP and UTP sites, and between the ATP-binding site and the glutamine amide transfer domain. Replacement of the nearby, fully conserved Lys 297 by alanine did not affect NH3-dependent CTP formation, relative to wild-type CTPS, but reduced k(cat) for the glutaminase activity 78-fold. Our findings suggest that the conformational change associated with binding ATP may be transmitted through the L10-alpha11 structural unit (residues 297-312) and thereby mediate effects on the glutaminase activity of CTPS.     

Glutamate synthase. Properties of the glutamine-dependent activity.

Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.     

Epidermal growth factor-induced phosphoinositide hydrolysis in permeabilized 3T3 cells: lack of guanosine triphosphate dependence and inhibition by tyrosine-containing peptides.

The possible involvement of a stimulatory guanosine triphosphate (GTP)-binding (G) protein in epidermal growth factor (EGF)-induced phosphoinositide hydrolysis has been investigated in permeabilized NIH-3T3 cells expressing the human EGF receptor. The mitogenic phospholipid lysophosphatidate (LPA), a potent inducer of phosphoinositide hydrolysis, was used as a control stimulus. In intact cells, pertussis toxin partially inhibits the LPA-induced formation of inositol phosphates, but has no effect on the response to EGF. In cells permeabilized with streptolysin-O, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) dramatically increases the initial rate of inositol phosphate formation induced by LPA. In contrast, activation of phospholipase C (PLC) by EGF occurs in a GTP-independent manner. Guanine 5'-O-(2-thiodiphosphate) (GDP beta S) which keeps G proteins in their inactive state, blocks the stimulation by LPA and GTP gamma S, but fails to affect the EGF-induced response. Tyrosine-containing substrate peptides, when added to permeabilized cells, inhibit EGF-induced phosphoinositide hydrolysis without interfering with the response to LPA and GTP gamma S. These data suggest that the EGF receptor does not utilize an intermediary G protein to activate PLC and that receptor-mediated activation of effector systems can be inhibited by exogenous substrate peptides.     

Characterization of the glutamine site of Escherichia coli guanosine 5'-monophosphate synthetase.

Alkylation of guanosine 5'-monophosphate (GMP) synthetase with the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid (chloroketon) and 6-diazo-5-oxonorleucine (DON) inactivated glutamine- and NH3-dependent GMP synthetase. Inactivation exhibited second order kinetics. Complete inactivation was accompanied by covalent attachment of 0.4 to 0.5 equivalent of chloroketon/subunit. Alkylation of GMP synthetase with iodacetamide selectively inactivated glutamine-dependent activity. The NH3-dependent activity was relatively unaffected. Approximately 1 equivalent of carboxamidomethyl group was incorporated per subunit. Carboxymethylcysteine was the only modified amino acid hydrolysis. Prior treatment with chloroketone decreased the capacity for alkylation by iodacetamide, suggesting that both reagents alkylate the same residue. GMP synthetase exhibits glutaminase activity when ATP is replaced by adenosine plus PPi. Iodoacetamide inactivates glutaminase concomitant with glutamine-dependent GMP synthetase. Analysis of pH versus velocity and Km data indicates that the amide of glutamine remains enzyme bound and does not mix with exogenous NH3 in the synthesis of GMP.     

Subunit interactions and glutamine utilization by Escherichia coli imidazole glycerol phosphate synthase

A selection strategy has been developed to identify amino acid residues involved in subunit interactions that coordinate the two half-reactions catalyzed by glutamine amidotransferases. The protein structures known for this class of enzymes have revealed that ammonia is shuttled over long distances and that each amidotransferase evolved different molecular tunnels for this purpose. The heterodimeric Escherichia coli imidazole glycerol phosphate (IGP) synthase was probed to assess if residues in the substrate amination subunit (HisF) are critical for the glutaminase activity in the HisH subunit. The activity of the HisH subunit is dependent upon binding of the nucleotide substrate at the HisF active site. This regulatory function has been exploited as a biochemical selection of mutant HisF subunits that retain full activity with ammonia as a substrate but, when constituted as a holoenzyme with wild-type HisH, impair the glutamine-dependent activity of IGP synthase. The steady-state kinetic constants for these IGP synthases with HisF alleles showed three distinct effects depending upon the site of mutation. For example, mutation of the R5 residue has similar effects on the glutamine-dependent amidotransfer reaction; however, k(cat)/K(m) for the glutaminase half-reaction was increased 10-fold over that for the wild-type enzyme with nucleotide substrate. This site appears essential for coupling of the glutamine hydrolysis and ammonia transfer steps and is the first example of a site remote to the catalytic triad that modulates the process. The results are discussed in the context of recent X-ray crystal structures of glutamine amidotransferases that relate the glutamine binding and acceptor binding sites.     

Mutational analysis of conserved glycine residues 142, 143 and 146 reveals Gly(142) is critical for tetramerization of CTP synthase from Escherichia coli

CTPS (cytidine 5'-triphosphate synthase) catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the nitrogen source. Binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers. In the present study, site-directed mutagenesis was used to replace the fully conserved glycine residues 142 and 143 within the UTP-binding site and 146 within the CTP-binding site of Escherchia coli CTPS. CD spectral analyses of wild-type CTPS and the glycine mutants showed a slight reduction of approximately 15% in alpha-helical content for G142A and G143A relative to G146A and wild-type CTPS, suggesting some local alterations in structure. Relative to wild-type CTPS, the values of k(cat)/K(m) for ammonia-dependent and glutamine-dependent CTP formation catalysed by G143A were reduced 22- and 16-fold respectively, whereas the corresponding values for G146A were reduced only 1.4- and 1.8-fold respectively. The glutaminase activity (k(cat)) of G146A was similar to that exhibited by the wild-type enzyme, whereas that of G143A was reduced 7.5-fold. G146A exhibited substrate inhibition at high concentrations of ammonia and a partial uncoupling of glutamine hydrolysis from CTP production. Although the apparent affinity (1/[S](0.5)) of G143A and G146A for UTP was reduced approximately 4-fold, G146A exhibited increased co-operativity with respect to UTP. Thus mutations in the CTP-binding site can affect UTP-dependent activity. Surprisingly, G142A was inactive with both ammonia and glutamine as substrates. Gel-filtration HPLC experiments revealed that both G143A and G146A were able to form active tetramers in the presence of ATP and UTP; however, nucleotide-dependent tetramerization of G142A was significantly impaired. Our observations highlight the sensitivity of the structure of CTPS to mutations in the UTP- and CTP-binding sites, with Gly(142) being critical for nucleotide-dependent oligomerization of CTPS to active tetramers. This 'structural sensitivity' may limit the number and/or types of mutations that could be selected for during the development of resistance to cytotoxic pyrimidine nucleotide analogues.     

Intrinsic DNA-dependent ATPase activity of reverse gyrase

Reverse gyrase is a type I DNA topoisomerase that promotes positive supercoiling of closed-circular double-stranded DNA through an ATP-dependent reaction, and it was purified from an archaebacterium, Sulfolobus. When ATP is replaced by UTP, GTP, or CTP, this enzyme just relaxes the negatively supercoiled closed-circular double-stranded DNA. We found that reverse gyrase hydrolyzes ATP through a double-stranded DNA-dependent reaction. The superhelicity of the DNA did not affect the ATPase activity. However, reverse gyrase does not hydrolyze UTP, GTP, or CTP. Therefore, any of the four nucleotide 5'-triphosphates acts as an effector for the topoisomerase activity of reverse gyrase, but only ATP supports the positive supercoiling of closed-circular double-stranded DNA, through the energy released on its hydrolysis. Single-stranded DNA was a much more potent cofactor for the ATPase activity of the enzyme than double-stranded DNA, and it acted as a potent inhibitor for the topoisomerase activity on double-stranded DNA. These results indicate that reverse gyrase has higher affinity to single-stranded DNA than to double-stranded DNA, which suggests a cellular function of the enzyme.     

Nanometer propagation of millisecond motions in V-type allostery

Imidazole glycerol phosphate synthase (IGPS) is a V-type allosteric enzyme, which is catalytically inactive for glutamine hydrolysis until the allosteric effector, N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) binds 30?? away. In the apo state, NMR relaxation dispersion experiments indicate the absence of millisecond (ms) timescale motions. Binding of the PRFAR to form the active ternary complex is endothermic with a large positive entropy change. In addition, there is a protein wide enhancement of conformational motions in the ternary complex, which connect the two active sites. NMR chemical shift changes and acrylamide quenching experiments suggest that little in the way of structural changes accompany these motions. The data indicate that enzyme activation in the ternary complex is primarily due to an enhancement of ms motions that allows formation of a population of enzymatically active conformers.