Adenosine 5'-triphosphate analogues as structural probes for Escherichia coli glutamine synthetase

Introduction of specific structural probes into substrate binding sites of Escherichia coli glutamine synthetase is now possible. Various analogues of ATP substituted with an amino or sulfhydryl moiety at the 6- or 8-position of the purine ring have been found to substitute for ATP in the autoinactivation reaction of the manganese enzyme with L-Met-(S)-sulfoximine at pH 7. Dissociation of enzyme complexes containing an ADP analogue, L-Met-(S)-sulfoximine phosphate, and 2 equiv of Mn2+ is negligible at neutral pH. Prior to binding of the mercapto nucleotides to active sites, 6-mercaptopurine ribonucleoside triphosphate (6-S-ATP) and 8-mercaptoadenosine 5'-triphosphate (8-S-ATP) also have been further modified with fluorescent and chromogenic probes for energy-transfer measurements [Maurizi, M. R., Kasprzyk, P. G., & Ginsburg, A. (1986) Biochemistry (following paper in this issue)] or with electron-dense markers for electron microscopic and X-ray crystallographic structural analyses. Binding 6-S-ATP or 8-S-ATP to enzyme active sites at pH 7.1 produced red shifts of approximately 6 nm in nucleotide spectra characteristic for transfer of these nucleotide analogues into more acidic and hydrophobic environments. The spectrum of 6-S-ADP at active sites was more red-shifted than that of 6-S-AMP attached to adenylylation sites. The thiol group at the 6- or 8-position of the purine ring of the bound nucleotides was accessible for reactions with alkylating or mercurial reagents. Alkylation or mercaptide formation produced large blue shifts in the spectrum of enzyme-bound 6-S-ADP or 8-S-ADP at active sites or of 6-S-AMP covalently bound at adenylylation sites. At least one of two tryptophanyl residues in each subunit is very near the nucleotide binding site, as evidenced by changes in tryptophanyl residue fluorescence on binding ATP, mercaptonucleotides, or other ATP analogues.     

Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase.

1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2'-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site. 4. The nucleotide specificities of 'coupled processes' nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-32Pi exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.     

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.     

Chloroplast F1 ATPase has more than three nucleotide binding sites, and 2-azido-ADP or 2-azido-ATP at both catalytic and noncatalytic sites labels the beta subunit

The photolabeling of chloroplast F1 ATPase, following exposure to Mg2+ and 2-azido-ATP and separation from medium nucleotides, results in derivatization of two separate peptide regions of the beta subunit. Up to 3 mol of the analogue can be incorporated per mole of CF1, with covalent binding of one moiety or two moieties per beta subunit that can be either AMP, ADP, or ATP derivatives. These results, the demonstration of noncovalent tight binding of at least four [3H]adenine nucleotides to the enzyme and the presence of three beta subunits per enzyme, point to six potential adenine nucleotide binding sites per molecule. The tightly bound 2-azido nucleotides on CF1, found after exposure of the heat-activated and EDTA-treated enzyme to Mg2+ and 2-azido-ATP, differ in their ease of replacement during subsequent hydrolysis of ATP. Some of the bound nucleotides are not readily replaced during catalytic turnover and covalently label one peptide region of the beta subunit. They are on noncatalytic sites. Other tightly bound nucleotides are readily replaced during catalytic turnover and label another peptide region of the beta subunit. They are at catalytic sites. No alpha-subunit labeling is detected upon photolysis of the bound 2-azido nucleotides. However, one or both of the sites could be at an alpha-beta-subunit interface with the 2-azido region close to the beta subunit, or both binding sites may be largely or entirely on the beta subunit.     

Effect of adenosine 5'-triphosphate and adenosine 5'-diphosphate on the oxidation of cytochrome c by cytochrome c oxidase

Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and inorganic pyrophosphate partially inhibit the oxidation of exogenous cytochrome c by cytochrome c oxidase of submitochondrial particles (with or without detergent treatment) or by a purified preparation when it is assayed polarographically in buffers of nonbinding ions at pH 7.8. ATP is somewhat more inhibitory than ADP. The inhibition is never greater than 50%, and it is always less than an equal concentration of Mg2+ ions is present or when the assays are run at pH 6. In contrast, the effect of ATP, ADP, and pyrophosphate on oxidase assays run spectrophotometrically is a similar slight stimulation of the oxidase of submitochondrial particles treated with deoxycholate and little or no effect on purified oxidase. The reaction of the oxidase of submitochondrial particles with the endogenous cytochrome c is stimulated by the nucleotides, as is the reduced nicotinamide adenine dinucleotide (NADH) oxidase activity. The observations can be explained by binding of ATP, ADP, or pyrophosphate to cytochrome c so that the formation of an especially reactive combination of cytochrome c and cytochrome oxidase previously postulated [Smith, L., Davies, H. C., & Nava, M. E. (1979) Biochemistry 18, 3140] is prevented. The data give no evidence that respiration via cytochrome c oxidase is regulated physiologically by direct effects of ATP or ADP on its activity.     

Purine but not pyrimidine nucleotides support rotation of F(1)-ATPase

The binding change model for the F(1)-ATPase predicts that its rotation is intimately correlated with the changes in the affinities of the three catalytic sites for nucleotides. If so, subtle differences in the nucleotide structure may have pronounced effects on rotation. Here we show by single-molecule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring in purine is indispensable for proper operation of this molecular motor. Although the three purine nucleotides were bound to the enzyme at different rates, all showed similar rotational characteristics: counterclockwise rotation, 120 degrees steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of approximately 40 piconewtons (pN).nm, and mechanical work done in a step of approximately 80 pN.nm. These latter characteristics are likely to be determined by the rotational mechanism built in the protein structure, which purine nucleotides can energize. With ATP and GTP, rotation was observed even when the free energy of hydrolysis was -80 pN.nm/molecule, indicating approximately 100% efficiency. Reconstituted F(o)F(1)-ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F(1) very slowly hydrolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.     

Nucleotide binding to isolated alpha and beta subunits of proton translocating adenosine triphosphatase studied with circular dichroism

The catalytic and allosteric sites of proton translocating adenosine triphosphatase (ATPase) were studied by measuring the binding of nucleotides to the ATPase, and its alpha and beta subunits purified from thermophilic bacterium PS3, with a circular dichroic spectrometer. In contrast to mesophilic ATPases, this thermophilic enzmye contained no tightly bound nucleotides, and its subunits were stable after their purification. These properties were advantageous for analyzing both catalytic and allosteric sites. The former site showed rapid and loose binding, but the latter slow (t 1/2 = 1 h, for ADP) and tight binding. When a nucleotide was bound, the beta subunits showed a negative ellipticity at 275 nm corresponding to a tyrosyl residue, while the alpha subunits showed an ellipticity change corresponding to the absorption curve of the bound nucleotide. This difference enabled us to distinguish the binding sites in ATPase. At a low concentration, ADP selectively bound to alpha subunits in the ATPase, while at a high concentration, it bound to both subunits. This finding suggests that the tight binding sites are located in the alpha subunits. Although ADP and ATP bound to both the purified alpha and beta subunits, CTP did not bind to beta but only to alpha subunits, and ITP bound to beta but hardly to alpha. These nucleotide specificities also supported the idea that the catalytic sites are located in the beta subunits and the allosteric sites are located in the alpha subunits.     

Catalysis of phosphoryl transfer from adenosine-5'-triphosphate (ATP) by trinuclear "chelate" complexes

The chelate ligand 2,9-di(6'-alpha-phenol-n-2',5'-diazahexyl)-1,10-phenanthroline (L) was synthesized and fully characterized. This ligand formed six protonated species in the solution. The bindings of the ligand to the nucleotide anions ATP, ADP and AMP were described in detail, with equilibrium constants given for each species formed. The strength of binding increased with the number of protons, corresponding to an increase in the number of hydrogen bonds and an increase in the coulombic attractive forces. At the same time, the coordination properties of the ternary complexes formed from the chelate ligand above, M (M=Zn(2+), Cd(2+)) and adenosine-5'-triphosphate (ATP) were studied. The metal complexes of the chelate recognize the nucleotides via multiple interactions similar to those occurring in the center of enzymes. The hydrolysis of ATP was studied with the mononuclear and trinuclear chelate complexes.     

Enzymatic synthesis of 5-azacytidine 5'-triphosphate from 5-azacytidine.

5-Azacytidine 5′-monophosphate (5-aza-CMP) was synthesized enzymatically from 5-azacytidine (5-aza-C) in a reaction catalyzed by uridine-cytidine kinase. In a second step, 5-azacytidine 5′-triphosphate (5-aza-CTP) was synthesized enzymatically from 5-aza-CMP using CMP kinase and nucleoside diphosphokinase. Due to the chemical instability of the triazide ring of 5-azacytosine at neutral and alkaline pH, the enzymatic synthesis and purification of the nucleotides by ion exchange chromatography were performed at acid pH. The enzymatically synthesized 5-aza-CTP had an ultraviolet absorbance spectrum at pH 5.5 similar to the spectrum of 5-aza-C. In the DNA-dependent RNA polymerase reaction, 5-aza-CTP inhibited the incorporation of [³H]CTP, but [³H]UTP, into RNA.     

Photoaffinity labeling of rabbit muscle fructose-1,6-bisphosphate aldolase with 8-azido-1,N6-ethenoadenosine 5'-triphosphate

Steady-state kinetic measurements have shown that 8-azido-1,N6-ethenoadenosine 5'-triphosphate (8-N3-epsilon ATP) can be noncovalently bound to rabbit muscle fructose 1,6-bisphosphate aldolase with Ki = 0.075 mM at pH 8.5. This binding is purely competitive with substrate and occurs at the strong binding site for mononucleotides. Photoaffinity labeling of aldolase in the presence of 8-azido-1,N6-ethenoadenosine 5'-triphosphate results in inactivation of the enzyme. Aldolase is protected against modification in the presence of the inhibitors hexitol 1,6-bisphosphate or ATP. The labeling is saturable, and a good correlation is observed between the loss of enzymatic activity and the incorporation of 8-N3-epsilon ATP into aldolase. In addition, aldolase loses its ability to bind to phosphocellulose following modification. Digestion of labeled protein with trypsin, chymotrypsin, and cyanogen bromide revealed substantial modification of peptide 259-269. Thr-265 was identified as the residue that was covalently modified by 8-N3-epsilon ATP. On the basis of these results and other data we propose a model for the mononucleotide binding site.     

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.     

Modulation by bicarbonate, phosphate, and maleate of the kinetics of adenosinetriphosphatase activity and of the binding of manganese ions to chloroplast coupling factor 1

Bicarbonate, maleate, and phosphate were shown to modulate adenosinetriphosphatase (ATPase) activity in coupling factor 1 from chloroplasts. Kinetic analysis of the changes in the ratio between the apparent Km with and without effectors indicated that the stimulation of the activity by bicarbonate was a result of a decrease in the Km for MnATP2-. The inhibition by phosphate resulted from a decrease in the Ki for free ATP as a competitive inhibitor at pH 8. THe effectors did not change Vmax at this pH. However, at pH 6.5, both Km and Vmax of ATPase activity with MnATP2- were changed by maleate, yet the mode of inhibition by free ATP remained unaltered. In addition to decreasing the Km, bicarbonate induced a 10-fold decrease in the Kd for binding of Mn2+ at the two tight binding sites in the presence of ATP at pH 8. At pH 6.5, maleate also decreased both the Km for MnATP2- and the Kd for Mn2+ binding. A decrease in the Km of a substrate induced by an effector is likely to be a result of a decrease in the binding constant of the substrate. Therefore, these results are in harmony with the suggested assignment of the two tight binding sites of Mn2+ at the active sites of the enzyme.     

Four tight nucleotide binding sites of chloroplast coupling factor 1.

We have examined the properties of the four tight nucleotide binding sites of reductively activated chloroplast coupling factor 1. Tight sites are here defined as those which retain bound nucleotides after passage of the chloroplast coupling factor 1 through Sephadex gel filtration centrifuge columns. Two of the sites, here called sites 4 and 5, have not been characterized in detail before. Site 4 has properties similar to those of site 1. It binds to ADP, ATP, and adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) tightly in the presence or absence of Mg2+. Bound ADP exchanges rapidly with medium ADP, but rapid exchange with ATP or AMP-PNP requires Mg2+. Site 4 may slowly hydrolyze bound ATP in the absence of medium nucleotides. Site 5 has properties similar to those of site 2. Tight binding of ATP and AMP-PNP requires Mg2+, but Mg29+)-ADP is not tightly bound. Site 5 does not hydrolyze bound ATP in the absence of medium nucleotides. Complete filling of all four tight nucleotide binding sites requires about one millimolar nucleotide, suggesting that low affinity binding sites are converted to tight binding via a nucleotide binding-induced conformational change.     

Properties of noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1

Nucleotide binding properties of two vacant noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1 (CF(1)) were studied. Kinetics of nucleotide binding to noncatalytic sites is described by the first-order equation that allows for two nucleotide binding sites that differ in kinetic features. Dependence of the nucleotide binding rate on nucleotide concentration suggests that tight nucleotide binding is preceded by rapid reversible binding of nucleotides. ADP binding is cooperative. The preincubation of CF(1) with Mg(2+) produces only slight effect on the rate of ADP binding and decreases the ATP binding rate. The ATP and ADP dissociation from noncatalytic sites is described by the first-order equation for similar sites with dissociation rate constants k(-2)(ADP)=1.5 x 10(-1) min(-1) and k(-2)(ATP) congruent with 10(-3) min(-1), respectively. As follows from the study, the noncatalytic sites of CF(1) are not homogeneous. One of them retains the major part of endogenous ADP after CF(1) precipitation with ammonium sulfate. Its other two sites can bind both ADP and ATP but have different kinetic parameters and different affinity for nucleotides.     

Interactions between the mitochondrial adenosinetriphosphatase and periodate-oxidized adenosine 5'-triphosphate, an affinity label for adenosine 5'-triphosphate binding sites

Periodate-oxidized ATP (o-ATP) was prepared as an affinity label of nucleotide binding sites on the chloroform-released ox heart mitochondrial ATPase. In the presence of MgSO4, o-ATP is a substrate for the ATPase. It can act as a reversible, competitive inhibitor of ATPase activity and can also induce an irreversible inhibition of ATPase activity. In parallel with the irreversible inhibition, covalent incorporation of [3H]o-ATP occurs. ATPase has about 1.05 mol of o-ATP bound per mol of ATPase when the enzyme is 50% inhibited. Most of the covalently bound o-ATP is associated with the alpha and beta subunits and is equally distributed between them. The incorporation of o-ATP into the ATPase is reduced, and the irreversible inhibition induced by o-ATP can be prevented totally by MgADP, MgATP, EDTA/ATP, or EDTA. The location, number, and the functional significance of the o-ATP binding sites are discussed. o-ATP can decompose to form an adenosine-containing compound and the tripolyphosphate anion in a beta-elimination reaction mechanism. The structures of the adenine-containing compound and its borohydride reduction product were determined. The adenine-containing elimination product inhibited the mitochondrial ATPase activity at a rate greater than that observed with o-ATP. The nature and mechanism of the inhibition of ATPase activity exerted by o-ATP and the elimination product were examined. The significance of the beta-elimination reaction to the use of periodate-oxidized nucleotides as affinity labels of nucleotide binding sites on other proteins is discussed.     

Inactivation and covalent modification of CTP synthetase by thiourea dioxide.

Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues.     

NMR of a synthetic peptide spanning the triphosphate binding site of adenosine 5'-triphosphate in actin

The amino acid residues 114-118 in actin were found to be implicated strongly in the binding of nucleotide, and as would be expected for such an important binding site, they are located in a completely conserved region of the actin sequence. A 19-residue peptide with the actin sequence 106-124 was synthesized in order to span the putative triphosphate binding site. Proton NMR spectra of the actin peptide 114-118 in the presence and absence of ATP indicated that Arg-116 and Lys-118 are particularly involved in binding ATP. A strong binding of ATP to the peptide 106-124 also was measured. Tripolyphosphate bound to the peptide 106-124 somewhat more weakly than ATP. Binding involved residues 115-118 and 121-124, indicating the presence of a reverse turn between these segments. Proton resonances were assigned by using two-dimensional double quantum correlated spectroscopy, one-dimensional spin decoupling techniques, one-dimensional nuclear Overhauser enhancement difference spectroscopy, and pH titration. The alpha CH resonances of Ala-3 and Asn-6 are markedly shifted downfield with respect to values in small unstructured peptides due to their close proximity to the side chains of Pro-4 and Pro-7, respectively. Several other resonances display chemical shifts which are indicative of a structured environment. Assignment of the amide proton resonances in H2O and measurements of the coupling constant 3JHNCH and the chemical shifts of the amide protons reveal that much of the synthetic peptide, particularly the backbone, exhibits a highly structured environment and represents a good model for the triphosphate binding site in actin.     

The use of nucleotide analogs to evaluate the mechanism of the heterotropic response of Escherichia coli aspartate transcarbamoylase

As an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used. These nucleotide analogs have the advantage over site-specific mutagenesis experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function. The ATP analogs purine 5'-triphosphate (PTP), 6-chloropurine 5'-triphosphate (Cl-PTP), 6-mercaptopurine 5'-triphosphate (SH-PTP), 6-methylpurine 5'-triphosphate (Me-PTP), and 1-methyladenosine 5'-triphosphate (Me-ATP) were partially synthesized from their corresponding nucleosides. Kinetic analysis was performed on the wild-type enzyme in the presence of these ATP analogs along with GTP, ITP, and XTP. PTP, Cl-PTP, and SH-PTP each activate the enzyme at subsaturating concentrations of L-aspartate and saturating concentrations of carbamoyl phosphate, but not to the same extent as does ATP. These experiments suggest that the interaction between N6-amino group of ATP and the backbone of the regulatory chain is important for orienting the nucleotide and inducing the displacements of the regulatory chain backbone necessary for initiation of the regulatory response. Me-PTP and Me-ATP also activate the enzyme, but in a more complex fashion, which suggests differential binding at the two sites within each regulatory dimer. The purine nucleotides GTP, ITP, and XTP each inhibit the enzyme but to a lesser extent than CTP. The influence of deoxy and dideoxynucleotides on the activity of the enzyme was also investigated. These experiments suggest that the 2' and 3' ribose hydroxyl groups are not of significant importance for binding and orientation of the nucleotide in the regulatory binding site. 2'-dCTP inhibits the enzyme to the same extent as CTP, indicating that the interactions of the enzyme to the O2-carbonyl of CTP are critical for CTP binding, inhibition, and the ability of the enzyme to discriminate between ATP and CTP. Examination of the electrostatic surface potential of the nucleotides and the regulatory chain suggest that the complimentary electrostatic interactions between the nucleotides and the regulatory chain are important for binding and orientation of the nucleotide necessary to induce the local conformational changes that propagate the heterotropic effect.     

Divergent evolutions of trinucleotide polymerization revealed by an archaeal CCA-adding enzyme structure

CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase], a template-independent RNA polymerase, adds the defined 'cytidine-cytidine-adenosine' sequence onto the 3' end of tRNA. The archaeal CCA-adding enzyme (class I) and eubacterial/eukaryotic CCA-adding enzyme (class II) show little amino acid sequence homology, but catalyze the same reaction in a defined fashion. Here, we present the crystal structures of the class I archaeal CCA-adding enzyme from Archaeoglobus fulgidus, and its complexes with CTP and ATP at 2.0, 2.0 and 2.7 A resolutions, respectively. The geometry of the catalytic carboxylates and the relative positions of CTP and ATP to a single catalytic site are well conserved in both classes of CCA-adding enzymes, whereas the overall architectures, except for the catalytic core, of the class I and class II CCA-adding enzymes are fundamentally different. Furthermore, the recognition mechanisms of substrate nucleotides and tRNA molecules are distinct between these two classes, suggesting that the catalytic domains of class I and class II enzymes share a common origin, and distinct substrate recognition domains have been appended to form the two presently divergent classes.