Catalytic domains of carbamyl phosphate synthetase. Glutamine-hydrolyzing site of Escherichia coli carbamyl phosphate synthetase

We present evidence that cysteine 269 of the small subunit of Escherichia coli carbamyl phosphate synthetase is essential for the hydrolysis of glutamine. When cysteine 269 is replaced with glycine or with serine by site-directed mutagenesis of the carA gene, the resulting enzymes are unable to catalyze carbamyl phosphate synthesis with glutamine as nitrogen donor. Even though the glycine 269, and particularly the serine 269 enzyme bind significant amounts of glutamine, neither glycine 269 nor serine 269 can hydrolyze glutamine. The mutations at cysteine 269 do not affect carbamyl phosphate synthesis with NH3 as substrate. The NH3-dependent activity of the mutant enzymes was equal to that of wild-type. Measurements of Km indicate that the enzyme uses unionized NH3 rather than ammonium ion as substrate. The apparent Km for NH3 of the wild-type enzyme is calculated to be about 5 mM, independent of pH. The substitution of cysteine 269 with glycine or with serine results in a decrease of the apparent Km value for NH3 from 5 mM with the wild-type to 3.9 mM with the glycine, and 2.9 mM with the serine enzyme. Neither the glycine nor the serine mutation at position 269 affects the ability of the enzyme to catalyze ATP synthesis from ADP and carbamyl phosphate. Allosteric properties of the large subunit are also unaffected. However, substitution of cysteine 269 with glycine or with serine causes an 8- and 18-fold stimulation of HCO-3 -dependent ATPase activity, respectively. The increase in ATPase activity and the decrease in apparent Km for NH3 provide additional evidence for an interaction of the glutamine binding domain of the small subunit with one of the two known ATP sites of the large subunit.     

Quantifying the allosteric properties of Escherichia coli carbamyl phosphate synthetase: determination of thermodynamic linked-function parameters in an ordered kinetic mechanism.

The effects of the allosteric ligands UMP, IMP, and ornithine on the partial reactions catalyzed by Escherichia coli carbamyl phosphate synthetase have been examined. Both of these reactions, a HCO3(-)-dependent ATP synthesis reaction and a carbamyl phosphate-dependent ATP synthesis reaction, follow bimolecular ordered sequential kinetic mechanisms. In the ATPase reaction, MgATP binds before HCO3- as established previously for the overall reaction catalyzed by carbamyl phosphate synthetase [Raushel, F. M., Anderson, P. M., & Villafranca, J. J. (1978) Biochemistry 17, 5587-5591]. The initial velocity kinetics for the ATP synthesis reaction indicate that MgADP binds before carbamyl phosphate in an equilibrium ordered mechanism except in the presence of ornithine. Determination of true thermodynamic linked-function parameters describing the impact of allosteric ligands on the binding interactions of the first substrate to bind in an ordered mechanism requires experiments to be performed in which both substrates are varied even if only one is apparently affected by the allosteric ligands. In so doing, we have found that IMP has little effect on the overall reaction of either of these two partial reactions. UMP and ornithine, which have a pronounced effect on the apparent Km for MgATP in the overall reaction, both substantially change the thermodynamic dissociation constant for MgADP from the binary E-MgADP complex, Kia, in the ATP synthesis reaction, with UMP increasing Kia 15-fold and ornithine decreasing Kia by 18-fold. By contrast, only UMP substantially affects the Kia for MgATP in the ATPase reaction, increasing it by 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of the reaction catalyzed by carbamyl phosphate synthetase. Binding of ATP to the two functionally different ATP sites.

Application of the pulse-chase procedure to study of the binding and utilization of ATP by glutamine-dependent carbamyl phosphate synthetase from Escherichia coli showed that the enzyme binds the two molecules of ATP used in this reaction at the same time, and that the two ATP-binding sites are functionally different. Thus, ATP bound to the first ATP site is used for carboxy phosphate formation, and ATP bound to the second ATP site is used for phosphorylation of carbamate. The present and previous findings support a mechanism that involves intermediate formation of two highly unstable intermediates: carboxy phosphate and carbamate. It is proposed that the presence of all of the reactants on the enzyme at the start of the catalytic cycle allows immediate utilization of these labile compounds in the carbamyl phosphate synthesis reaction.     

Carbamyl phosphate synthetase of Escherichia coli uses the same diastereomer of adenosine-5'-[2-thiotriphosphate] at both ATP sites.

Carbamyl phosphate synthetase from Escherichia coli has been shown to use only the A isomer of adenosine-5'-[2-thiotriphosphate] in both the ATPase reaction (MgATP HCO3- leads to MgADP + Pi) and the carbamyl phosphate synthesis reaction (2MgATP + HCO3- + L-glutamine leads to 2MgADP + Pi + carbamyl-P + L-glutamate). The B isomer was less than 5% as reactive. In the reverse reaction, only the A isomer of adenosine-5'-[2-thiotriphosphate] is synthesized from adenosine-5'-[2-thiodiphosphate] and carbamyl-P as determined by 31P NMR and a coupled enzymatic assay with Cd2+- hexokinase. It is therefore proposed that carbamyl phosphate synthetase uses the same diastereomer of MgATP at both ATP sites.     

Phosphorylation and activation of hamster carbamyl phosphate synthetase II by cAMP-dependent protein kinase. A novel mechanism for regulation of pyrimidine nucleotide biosynthesis.

The trifunctional protein CAD, which contains the first three enzyme activities of pyrimidine nucleotide biosynthesis (carbamyl phosphate synthetase II, aspartate transcarbamylase and dihydro-orotase), is phosphorylated stoichiometrically by cyclic AMP-dependent protein kinase. Phosphorylation activates the ammonia-dependent carbamyl phosphate synthetase activity of the complex by reducing the apparent Km for ATP. This effect is particularly marked in the presence of the allosteric feedback inhibitor, UTP, when the apparent Km is reduced by greater than 4-fold. Inhibition by physiological concentrations of UTP is substantially relieved by phosphorylation. Cyclic AMP-dependent protein kinase phosphorylates two serine residues on the protein termed sites 1 and 2, and the primary structures of tryptic peptides containing these sites have been determined: Site 1: Arg-Leu-Ser(P)-Ser-Phe-Val-Thr-Lys Site 2: Ile-His-Arg-Ala-Ser(P)-Asp-Pro-Gly-Leu-Pro-Ala-Glu-Glu-Pro-Lys During the phosphorylation reaction, activation of the carbamyl phosphate synthetase shows a better correlation with occupancy of site 1 rather than site 2. Both phosphorylation and activation can be reversed using purified preparations of the catalytic subunits of protein phosphatases 1- and -2A, and inactivation also correlates better with dephosphorylation of site 1 rather than site 2. We believe this to be the first report that a key enzyme in nucleotide biosynthesis is regulated in a significant manner by reversible covalent modification. The physiological role of this phosphorylation in the stimulation of cell proliferation by growth factors and other mitogens is discussed.     

Dissection of the functional domains of Escherichia coli carbamoyl phosphate synthetase by site-directed mutagenesis

The catalytic functions of the amino-terminal and carboxyl-terminal halves of the large subunit of carbamoyl phosphate synthetase from Escherichia coli have been identified using site-directed mutagenesis. Glycine residues at positions 176, 180, and 722 within the putative mononucleotide-binding site were replaced with isoleucine residues. Each of these mutations resulted in at least a 1 order of magnitude reduction in the Vmax for carbamoyl phosphate synthesis. The mutations on the amino-terminal half, G176I and G180I, caused slight reduction in the rate of synthesis of ATP from ADP and carbamoyl phosphate (the partial ATP synthesis reaction) but the bicarbonate-dependent ATPase reaction velocity was reduced to less than 10% of the wild-type rate. The mutant G722I, which is on the carboxy-terminal half, caused the partial ATP synthesis reaction to be reduced by 1 order of magnitude but the bicarbonate-dependent ATPase reaction was reduced only slightly. All three mutations are within regions which show homology to the putative glycine-rich loops of many ATP-binding proteins. These results have been interpreted to suggest that the two homologous halves of the large subunit of carbamoyl phosphate synthetase each contain a binding site for ATP. The NH2-terminal domain contains the portion of the large subunit that is primarily involved with the phosphorylation of bicarbonate to carboxy phosphate while the COOH-terminal domain contains the region of the enzyme that catalyzes the phosphorylation of carbamate to carbamoyl phosphate.     

Mitochondrial carbamoyl phosphate synthetase activity in the absence of N-acetyl-L-glutamate. Mechanism of activation by this cofactor

Rat liver carbamoyl-phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. Km values for ATP, Mg2+ and K+ are greatly increased, the Km for HCO-3 is not changed much, and the Km for NH+4 is markedly reduced. Vmax for the synthetase reaction is less than 20% of that of the acetylglutamate-activated enzyme whereas Vmax for the ATPase activity is greater than 40% of that with acetylglutamate. Pulse-chase experiments with H14CO-3 show formation of less "active CO2" (the central intermediate) than with acetylglutamate; ATPase activity is reduced in proportion, but the synthetase activity is much smaller. Binding of one ATP molecule with high affinity (Kd = 20-30 microM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATPB (ATPB provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATPA (ATPA yields Pi) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 microM acetylglutamate the lag is much longer, but at 10 mM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATPA and the ATPB binding sites. Preincubation with 10 mM acetylglutamate alone shortens the activation time. From these findings we propose an allosteric model for activation of carbamoyl-phosphate synthetase in which there are two active states, R and R . AcGlu. Binding of ATPA is associated with the conversion of T to R. R . AcGlu differs from R in that transfer to carbamate of the gamma-phosphoryl group of ATPB appears to be facilitated.     

A mutation that uncouples allosteric regulation of carbamyl phosphate synthetase in Drosophila

In animals, UTP feedback inhibition of carbamyl phosphate synthetase II (CPSase) controls pyrimidine biosynthesis. Suppressor of black (Su(b) or rSu(b)) mutants of Drosophila melanogaster have elevated pyrimidine pools, and this mutation has been mapped to the rudimentary locus. We report that rSu(b) is a missense mutation resulting in a glutamate to lysine substitution within the second ATP binding site (i.e. CPS.B2 domain) of CPSase. This residue corresponds to Glu780 in the Escherichia coli enzyme (Glu1153 in hamster CAD) and is universally conserved among CPSases. When a transgene expressing the Glu-->Lys substitution was introduced into Drosophila lines homozygous for the black mutation, the resulting flies exhibited the Su(b) phenotype. Partially purified CPSase from rSu(b) and transgenic flies carrying this substitution exhibited a dramatic reduction in UTP feedback inhibition. The slight UTP inhibition observed with the Su(b) enzyme in vitro was due mainly to chelation of Mg2+ by UTP. However, the Km values for glutamate, bicarbonate, and ATP obtained from the Su(b) enzyme were not significantly different from wild-type values. From these experiments, we conclude that this residue plays an essential role in the UTP allosteric response, probably in propagating the response between the effector binding site and the ATP binding site. This is the first CPSase mutation found to abolish feedback inhibition without significantly affecting other enzyme catalytic parameters.     

Carbamoyl-phosphate synthetase II of the mammalian CAD protein: kinetic mechanism and elucidation of reaction intermediates by positional isotope exchange

The kinetic mechanism of carbamoyl-phosphate synthetase II from Syrian hamster kidney cells has been determined at pH 7.2 and 37 degrees C. Initial velocity, product inhibition, and dead-end inhibition studies of both the biosynthetic and bicarbonate-dependent adenosinetriphosphatase (ATPase) reactions are consistent with a partially random sequential mechanism in which the ordered addition of MgATP, HCO3-, and glutamine is followed by the ordered release of glutamate and Pi. Subsequently, the binding of a second MgATP is followed by the release of MgADP, which precedes the random release of carbamoyl phosphate and a second MgADP. Carbamoyl-phosphate synthetase II catalyzes beta gamma-bridge:beta-nonbridge positional oxygen exchange of [gamma-18O]ATP in both the ATPase and biosynthetic reactions. Negligible exchange is observed in the strict absence of HCO3- (and glutamine or NH4+). The ratio of moles of MgATP exchanged to moles of MgATP hydrolyzed (nu ex/nu cat) is 0.62 for the ATPase reaction, and it is 0.39 and 0.16 for the biosynthetic reaction in the presence of high levels of glutamine and NH4+, respectively. The observed positional isotope exchange is suppressed but not eliminated at nearly saturating concentrations of either glutamine or NH4+, suggesting that this residual exchange results from either the facile reversal of an E-MgADP-carboxyphosphate-Gln(NH4+) complex or exchange within an E-MgADP-carbamoyl phosphate-MgADP complex, or both. In the 31P NMR spectra of the exchanged [gamma-18O]ATP, the distribution patterns of 16O in the gamma-phosphorus resonances in all samples reflect an exchange mechanism in which a rotationally unhindered molecule of [18O3, 16O]Pi does not readily participate. These results suggest that the formation of carbamate from MgATP, HCO3-, and glutamine proceeds via a stepwise, not concerted mechanism, involving at least one kinetically competent covalent intermediate, such as carboxyphosphate.     

A new approach of the estimation of Km of carbamyl phosphate synthetase for ammonia in isolated rat hepatocytes

1. The Km for ammonia of carbamyl phosphate synthetase was determined by preincubating isolated liver cells for 30 min in the absence of ammonia and bicarbonate and in the presence of ornithine, chloroquine, which blocks lysosomal proteolysis, and aminoxy acetic acid, which inhibits transaminases. 2. The reaction was started with the addition of varying concentrations of ammonia and 10 mM bicarbonate. 3. The rate of citrulline formation was measured as related to ammonia concentration. 4. The pre-incubation with ornithine permits an accumulation of intracellular and mitochondrial ornithine concentrations which in turn allow rapid citrulline formation in the carbamyl phosphate form. 5. This prevents any feedback inhibition on a carbamyl phosphate synthetase or decreases in activity due to accumulation of carbamyl phosphate and/or absence of ornithine. 6. Using these methods in combination with [14C]bicarbonate permitted an estimation of exogenous ammonia for carbamyl phosphate synthesis. 7. The Km for ammonia was 1.5 mM, using a pK of 8.88 the Km for free NH3 was 48 microM.     

In vivo synthesis of carbamyl phosphate from NH3 by the large subunit of Escherichia coli carbamyl phosphate synthetase

The cloned carAB operon of Escherichia coli coding for the small and large subunits of carbamyl phosphate synthetase has been used to construct a recombinant plasmid with a 4.16 kilobase ClaI fragment of the car operon that lacks the major promoters, P1 and P2. The plasmid, pHN12, carries a functional carB gene. A mutant E. coli strain lacking both subunits of carbamyl phosphate synthetase when transformed with pHN12 overproduces the large subunit by 200-fold (8-10% of the cellular protein). The elevated levels of the large subunit enable the transformed cells to utilize NH3 but not glutamine as nitrogen donor for carbamyl phosphate synthesis. The large subunit has been purified from the overexpressing strain. The purified native large subunit is capable of synthesizing carbamyl phosphate from ammonia, HCO-3, and ATP. The kinetic properties of the large subunit compared with the holoenzyme indicate that the Michaelis constants of the large subunit for HCO-3 and ATP are modulated by its association with the small glutamine binding subunit.     

15N isotope effects in glutamine hydrolysis catalyzed by carbamyl phosphate synthetase: evidence for a tetrahedral intermediate in the mechanism

15N isotope effects have been measured on the hydrolysis of glutamine catalyzed by carbamyl phosphate synthetase of Escherichia coli. The isotope effect in the amide nitrogen of glutamine is 1. 0217 at 37 degrees C with the wild-type enzyme in the presence of MgATP and HCO(3)(-) (overall reaction taking place). This V/K isotope effect indicates that breakdown of the tetrahedral intermediate formed with Cys 269 to release ammonia is the rate-limiting step in the hydrolysis. A full isotope effect of 1. 0215 is also seen in the partial reaction catalyzed by an E841K mutant enzyme, whose rate of glutamine hydrolysis is not affected by MgATP and HCO(3)(-). With wild-type enzyme in the absence of MgATP and HCO(3)(-), however, the (15)N isotope effect is reduced to 1. 0157. These isotope effects are interpreted in terms of partitioning of the tetrahedral intermediate whose rate of formation is dependent upon a conformation change which closes the active site after glutamine binding and prepares the enzyme for catalysis. An Ordered Uni Bi mechanism for glutamine hydrolysis that is consistent with the isotope effects and with the catalytic properties of the enzyme is proposed.     

Identification of the ATP binding sites of the carbamyl phosphate synthetase domain of the Syrian hamster multifunctional protein CAD by affinity labeling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) was used to chemically modify the ATP binding sites of the carbamyl phosphate synthetase domain of CAD, the multifunctional protein that catalyzes the first steps in mammalian pyrimidine biosynthesis. Reaction of CAD with FSBA resulted in the inactivation of the ammonia- and glutamine-dependent CPSase activities but had no effect on its glutaminase, aspartate transcarbamylase, or dihydroorotase activities. ATP protected CAD against inactivation by FSBA whereas the presence of the allosteric effectors UTP and PRPP afforded little protection, which suggests that the ATP binding sites were specifically labeled. The inactivation exhibited saturation behavior with respect to FSBA with a K1 of 0.93 mM. Of the two ATP-dependent partial activities of carbamyl phosphate synthetase, bicarbonate-dependent ATPase was inactivated more rapidly than the carbamyl phosphate dependent ATP synthetase, which indicates that these partial reactions occur at distinct ATP binding sites. The stoichiometry of [14C]FSBA labeling showed that only 0.4-0.5 mol of FSBA/mol of protein was required for complete inactivation. Incorporation of radiolabeled FSBA into CAD and subsequent proteolysis, gel electrophoresis, and fluorography demonstrated that only the carbamyl phosphate synthetase domain of CAD is labeled. Amino acid sequencing of the principal peaks resulting from tryptic digests of FSBA-modified CAD located the sites of FSBA modification in regions that exhibit high homology to ATP binding sites of other known proteins. Thus CAD has two ATP binding sites, one in each of the two highly homologous halves of the carbamyl phosphate domain which catalyze distinct ATP-dependent partial reactions in carbamyl phosphate synthesis.     

Mechanism of mitochondrial carbamoyl-phosphate synthetase: synthesis and properties of active CO2, precursor of carbamoyl phosphate.

This paper demonstrates the formation of "active CO2" (CO2-P), a precursor of carbamoyl phosphate (CP), with frog liver carbamoyl-phosphate synthetase. Absence of ammonia is essential for the demonstration by pulse incubation with H14CO3- of CO2-P. Adenosine triphosphate (ATP) and acetylglutamate are required for the synthesis of CO2-P, which is highly unstable in aqueous solutions (t1/2 = 0.75 s at 24 degrees C at neutral pH). In the absence of ammonia, CO2-P attains rapidly a steady-state level, which depends on the concentration of ATP and HCO3-. The "apparent KM'S" are approximately equal to those found for the adenosine triphosphate (ATPase) activity of the enzyme. The maximum level of CO2-P is limited by the amount of enzyme, and approximates 4 mol of intermediate/mol of enzyme. The unprotonated form of ammonia seems to be the species reacting with CO2-P to produce CP. The reaction of CO2-P and NH3 is very fast (rate constant kn = 8 x 10(4) M-1 S-1) and does not consume free ATP. Therefore, the 2 mol of ATP necessary for CP synthesis binds or reacts with the enzyme and/or CO2 prior to reaction with NH3. The reaction of CO2-P with NH3 also takes place in acetone under conditions at which the enzyme is not active, suggesting little or no assistance from enzyme catalysis or that a part of the catalytic site is "frozen" by the solvent in the active conformation. In the light of these and other findings, a new scheme is proposed for the mechanism of frog liver carbamoyl-phosphate synthetase and some considerations are made on the chemical nature of the intermediate and on the possible evolutionary significance of the reaction of CO2-P with NH3 in acetone.     

Functional linkage between the glutaminase and synthetase domains of carbamoyl-phosphate synthetase. Role of serine 44 in carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (cad).

Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO(3)(-), the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser(44) in the GLN attenuation domain with alanine increases the k(cat)/K(m) for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser(44) GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO(3)(-) did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser(44) --> Ala mutant than that needed for carbamoyl phosphate synthesis. Ser(44), the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis.     

Carbamyl phosphate-dependent ATP synthesis catalyzed by formyltetrahydrofolate synthetase.

Formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) from Clostridium cylindrosporum catalyzes phosphate transfer from carbamyl phosphate to ADP. This activity is lost when monovalent cations are removed and is recovered when K+ is added back. Carbamyl phosphate is an inhibitor of the formyltetrahydrolfolate synthetase forward reaction, and formate as well as phosphate inhibit the ATP synthesis reaction. Acetyl phosphate and phosphonoacetate are inhibitors of both reactions. The results of kinetic studies support the concept that carbamyl phosphate is an analog of the putative intermediate of the formyltetrahydrofolate synthetase reaction, formyl phosphate.     

Transition state stabilization by a phylogenetically conserved tyrosine residue in methionyl-tRNA synthetase.

The crystal structure of a fully biologically active monomeric form of Escherichia coli methionyl-tRNA synthetase (MetRS) complexed with ATP has recently been reported (Brunie, S., Zelwer, C., and Risler, J.-L., (1990) J. Mol. Biol. 216, 411-424), revealing details of the active site of the enzyme, including the location of amino acid residues potentially involved in substrate binding. In the present paper, the role of 3 active site residues in interaction with methionine, ATP, and tRNA(fMet) and in catalysis of methionyl-adenylate has been explored using site-directed mutagenesis. Lys142 is located near the ribose of ATP in the MetRS.ATP cocrystal. Mutation of this residue to Ala caused a 5-fold decrease in kcat/Km for ATP-PPi exchange, indicating some contribution of the lysine side chain to the specificity of the enzyme. Mutation of Tyr359 to Ala produced a 14-fold increase in the Km for ATP with only a small (2-3-fold) change in the other kinetic parameters, indicating that the major role of this residue is in formation of the initial complex with ATP and/or in stabilization of the methionyl-adenylate reaction intermediate. Mutation of the adjacent residue Tyr358 to Ala had no effect on the Km values for methionine or ATP but produced nearly a 2000-fold decrease in the rate of ATP-PPi exchange. This mutation also dramatically reduced the rate of pyrophosphorolysis of the isolated MetRS.Met-AMP complex on addition of pyrophosphate without increasing the Km for PPi. None of the mutations affected the Km for tRNAfMet in the aminoacylation reaction. The results suggest that Tyr358 may enhance the rate of methionyl-adenylate formation by binding to the alpha-phosphate of ATP in the transition state. Interaction of Tyr358 and Tyr359 with ATP during the course of the reaction requires a significant change in the conformation of this region of the active site compared to the structure found in the MetRS.ATP complex. Such a shift is consistent with an induced-fit mechanism for methionine activation. Primary sequence comparisons of methionine-specific enzymes from yeast and bacterial sources reveals that Tyr358 is conserved in all of the known MetRS sequences.     

83-kilodalton heat shock proteins of trypanosomes are potent peptide-stimulated ATPases.

A Crithidia fasciculata 83-kDa protein purified during a separate study of C. fasciculata trypanothione synthetase was shown to have ATPase activity and to belong to the hsp90 family of stress proteins. Because no ATPase activity has previously been reported for the hsp90 class, ATP utilization by C. fasciculata hsp83 was characterized: this hsp83 has an ATPase kcat of 150 min-1 and a Km of 60 microM, whereas the homologous mammalian hsp90 binds ATP but has no ATPase activity. Crithidia fasciculata hsp83 undergoes autophosphorylation on serine and threonine at a rate constant of 3.3 x 10(-3) min-1. Similar analysis was performed on recombinant Trypanosoma cruzi hsp83, and comparable ATPase parameters were obtained (kcat = 100 min-1, Km = 80 microM, kautophosphorylation = 6.3 x 10(-3) min-1). The phosphoenzyme is neither on the ATPase hydrolytic pathway nor does it affect ATPase catalytic efficiency. Both C. fasciculata and T. cruzi hsp83 show up to fivefold stimulation of ATPase activity by peptides of 6-24 amino acids.     

Inhibition of carbamyl phosphate synthetase by P1, P5-di(adenosine 5')-pentaphosphate: evidence for two ATP binding sites.

Studies on the effect of a series of alpha, omega-diadenosine 5'-polyphosphate (ApnA; n = 2 to 6) on carbamyl phosphate synthetase showed that only Ap5A is an effective inhibitor. Ap5A also inhibits two partial reactions catalyzed by the enzyme: bicarbonate-dependent ATPase and ATP synthesis from carbamyl phosphate and ADP. The data indicate that Ap5A binds to the enzyme sites that interact with ATP. Of a variety of ATP-utilizing enzymes (kinases, hydrolases, synthetases), only adenylate kinase (Leinhard, G. E., and Secemski, I. I. (1973) J. Biol. Chem. 248, 1121--1123) and carbamyl phosphate synthetase are inhibited by Ap5A. The present findings provide strong evidence that carbamyl phosphate synthetase has two separate binding sites for ATP in which the gamma-phosphate moeities of ATP are bound in close proximity to the bicarbonate binding site of the enzyme.     

Structural defects within the carbamate tunnel of carbamoyl phosphate synthetase

The X-ray crystal structure of carbamoyl phosphate synthetase (CPS) from Escherichia coli has unveiled the existence of two molecular tunnels within the heterodimeric enzyme. These two interdomain tunnels connect the three distinct active sites within this remarkably complex protein and apparently function as conduits for the transport of unstable reaction intermediates between successive active sites. The operational significance of the ammonia tunnel for the migration of NH3 is supported experimentally by isotope competition and protein modification. The passage of carbamate through the carbamate tunnel has now been assessed by the insertion of site-directed structural blockages within this tunnel. Gln-22, Ala-23, and Gly-575 from the large subunit of CPS were substituted by mutagenesis with bulkier amino acids in an attempt to obstruct and/or hinder the passage of the unstable intermediate through the carbamate tunnel. The structurally modified proteins G575L, A23L/G575S, and A23L/G575L exhibited a substantially reduced rate of carbamoyl phosphate synthesis, but the rate of ATP turnover and glutamine hydrolysis was not significantly altered. These data are consistent with a model for the catalytic mechanism of CPS that requires the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain. The partial reactions of CPS have not been significantly impaired by these mutations, and thus, the catalytic machinery at the individual active sites has not been functionally perturbed.