CTP Binding Research Articles

Lysine-60 in the regulatory chain of Escherichia coli aspartate transcarbamoylase is important for the discrimination between CTP and ATP

Lysine-60 in the regulatory chain of aspartate transcarbamoylase has been changed to an alanine by site-specific mutagenesis. The resulting enzyme exhibits activity and homotropic cooperativity identical with those of the wild-type enzyme. The substrate concentration at half the maximal observed specific activity decreases from 13.3 mM for the wild-type enzyme to 9.6 mM for the mutant enzyme. ATP activates the mutant enzyme to the same extent that it does the wild-type enzyme, but the concentration of ATP required to reach half of the maximal activation is reduced approximately 5-fold for the mutant enzyme. CTP at a concentration of 10 mM does not inhibit the mutant enzyme, while under the same conditions CTP at concentrations less than 1 mM will inhibit the wild-type enzyme to the maximal extent. Higher concentrations of CTP result in some inhibition of the mutant enzyme that may be due either to hetertropic effects at the regulatory site or to competitive binding at the active site. UTP alone or in the presence of CTP has no effect on the mutant enzyme. Kinetic competition experiments indicate that CTP is still able to displace ATP from the regulatory sites of the mutant enzyme. Binding measurements by equilibrium dialysis were used to estimate a lower limit on the dissociation constant for CTP binding to the mutant enzyme (greater than 1 x 10(-3) M). Equilibrium competition binding experiments between ATP and CTP verified that CTP still can bind to the regulatory site of the enzyme. For the mutant enzyme, CTP affinity is reduced approximately 100-fold, while ATP affinity is increased by 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase.

The allosteric control of aspartate transcarbamoylase (ATCase, EC 2.1.3.2) of Escherichia coli involves feedback inhibition by both CTP and UTP rather than just CTP alone. It has been known that CTP functions as a heterotropic inhibitor of catalysis; however, the inhibition by CTP alone is incomplete (50-70% at various aspartate concentrations) and there is only a partial occupancy of the allosteric binding sites by CTP at saturating concentrations. The logic of these allosteric characteristics can now be understood in that UTP is a synergistic inhibitor of ATCase in the presence of CTP even though UTP has no independent effect at pH 7.0. When saturating concentrations of CTP are present, the concentration of substrate required for half-maximal activity (S0.5) of the native holoenzyme for aspartate increases from 5 to 11 mM. When CTP and UTP are both present, the aspartate requirement increases further (S0.5 = 17 mM). At aspartate concentrations less than 5 mM, the heterotropic inhibition of ATCase is 90-95% in the presence of both pyrimidine nucleotides. UTP does enhance the binding of CTP to the holoenzyme but the number of tight binding sites does not change (n = 3). The binding of UTP is stabilized in the presence of CTP although its binding characteristics are not as strong as those of CTP. The recent crystallographic studies of Kim et al. [Kim, H.K., Pan, Z., Honzatko, R.B., Ke, H.M. & Lipscomb, W.N. (1987) J. Mol. Biol. 196, 853-875] have described a structural asymmetry across the molecular two-fold axis that is consistent with these CTP/UTP interactions. The synergistic inhibition of ATCase by both CTP and UTP provides a satisfying logic for ensuring a balance of endogenous pyrimidine nucleotide pools.

Site-directed mutagenesis of a residue located in the regulatory site of Escherichia coli aspartate transcarbamoylase. Involvement of lysine 94 in effector binding and the allosteric mechanism.

Lysine 94 in the regulatory chain of aspartate transcarbamoylase has been changed to a glutamine residue by site-directed mutagenesis. The resulting enzyme is almost insensitive to the activator ATP and shows a substantially reduced response to the feedback inhibitor CTP. Competition experiments indicate that ATP is still able to bind at low concentrations to the regulatory site of the mutant enzyme, even though no stimulation could be detected. When the nucleosides adenosine or cytidine were used, the saturation curves of the mutant and the wild-type enzyme became indistinguishable. Together these results indicate that lysine 94 is strongly involved in the binding of ATP and CTP by interacting specifically with the triphosphate moiety of these nucleotide effectors. Furthermore, unlike the wild-type enzyme, the inhibitory and stimulatory effects in the mutant enzyme are insensitive to changes in aspartate concentrations, implying that the lysine 94 side chain is also involved in the allosteric mechanism of the enzyme.

Binding of CTP:phosphocholine cytidylyltransferase to large unilamellar vesicles

We have studied the binding of CTP:phosphocholine cytidylyltransferase from HeLa cell cytosol to large unilamellar vesicles of egg phosphatidylcholine (PC) or HeLa cell phospholipids that contain various amounts of oleic acid. A fatty acid/phospholipid molar ratio exceeding 10% was required for CTP:phosphocholine cytidylyltransferase binding to liposomes. At a fatty acid/phospholipid molar ratio of 1; 85% of the cytosolic CTP:phosphocholine cytidylyltransferase was bound. The enzyme also bound to liposomes with at least 20 mol% palmitic acid, monoolein, diolein or oleoylacetylglycerol. Oleoyl-CoA did not promote enzyme binding to liposomes. Binding to oleate-PC vesicles was blocked by Triton X-100 but not by 1 M KC1, and was reversed by incubation of the vesicles with bovine serum albumin. Cytidylyltransferase bound to egg PC vesicles that contained 33 mol% oleic acid equally well at 4 ° C and 37 ° C. The enzyme also bound to dimyristoyl- and dipalmitoylphosphatidylcholine vesicles containing oleic acid at temperatures below the phase transition for these liposomes. Binding of the cytidylyltransferase to egg PC vesicles containing oleic acid, monoolein, oleoylacetylglycerol or diolein resulted in enzyme activation, as did binding to dipalmitoylPC-oleic acid vesicles. However, binding to egg PC-palmitic acid vesicles did not fully activate the transferase. Various mechanisms for cytidylyltransferase interaction with membranes are discussed.

Communication between polypeptide chains in aspartate transcarbamoylase. Conformational changes at the active sites of unliganded chains resulting from ligand binding to other chains.

A largely inactive derivative of the catalytic subunit of Escherichia coli aspartate transcarbamoylase containing trinitrophenyl groups on lysine 83 and 84 was used to study communication between polypeptide chains in the holoenzyme and the isolated catalytic trimers. Addition of native regulatory dimers to the derivative yielded a holoenzyme-like complex of low activity which exhibited sigmoidal kinetics and was inhibited by CTP and activated by ATP. The binding of CTP and ATP to the regulatory subunits caused significant and opposite changes in the absorption spectrum resulting from changes in the environment of the sensitive chromophores at the active sites. In allosteric hybrid molecules containing one native and one trinitrophenylated catalytic subunit, along with native regulatory subunits, the binding of a bisubstrate analog, N-(phosphonacetyl)-L-aspartate, to the native catalytic subunit resulted in a perturbation of the spectrum of the chromophore on the unliganded modified chains. Thus the conformational changes associated with the allosteric transition responsible for both heterotropic and homotropic effects are propagated from the sites of ligand binding to the active sites of unliganded distant chains. In addition to the communication from regulatory chains to catalytic chains and the cross-talk from one catalytic subunit to the other, communication between individual catalytic chains in isolated trimers was also demonstrated. By constructing hybrid trimers containing one trinitrophenylated chain and two native chains, we could detect a change in the environment of the chromophore upon the binding of the bisubstrate analog to the native chains.

The stimulation and binding of CTP : Phosphorylcholine cytidylyltransferase by phosphatidylcholine-oleic acid vesicles

The activity of the low molecular weight form of cytidylyltransferase from fetal lung cytosol and adult liver cytosol was stimulated more by phosphatidylcholine-oleic acid (1:1 molar ratio) vesicles than by phosphatidylglycerol vesicles. Phosphatidylcholine alone did not stimulate the activity, while oleic acid alone produced only slight stimulation. Vesicles prepared from phosphatidylinositol, phosphatidylglycerol-cholesterol (2:1) and phosphatidylglycerol-phosphatidylcholine (1:1) all stimulated the activity to the same extent. Phosphatidylcholine-oleic acid vesicles (molar ratio 2:1) produced less stimulation than 1:1 vesicles. Phosphatidylcholine-palmitic acid vesicles (2:1) were about 50% as active as the corresponding phosphatidylcholine-oleic acid vesicles. All vesicles were in the size range of small unilamellar vesicles as judged by Sephacryl S-1000 chromatography. Stimulation also occurred when phosphatidylcholine vesicles and oleic acid were added separately to the assay. The stimulation by phospholipid vesicles was correlated with the ability of the vesicles to bind cytidylyltransferase, determined by sucrose density centrifugation of the enzyme-vesicles mixtures. We conclude that the stimulation of soluble cytidylyltransferase occurs through binding of the enzyme to anionic membrane surfaces. Suitable anionic membranes can be prepared either from anionic phospholipids, or by the addition of anionic lipids (unesterified fatty acids or phosphatidylglycerol) to phosphatidylcholine.

Interactions of phosphate ligands with Escherichia coli aspartate carbamoyltransferase in the crystalline state

We report here results of crystallographic studies at 3.0 Å resolution of complexes of phosphate ligands with aspartate carbamoyltransferase from Escherichia coli. Specifically, we interpret the binding of CTP, ATP, 5-bromo-CTP, 8-bromo-GTP. formycin A 5′-triphosphate. 3, N 6-etheno-ATP. phosphate/carbamoyl- d.l-aspartate and pyrophosphate to the catalytic and regulatory chains of the enzyme. We observed two modes of binding of ligands to the phosphate crevice of the catalytic chain. Pyrophosphate and phosphate penetrate deeply into the cleft between the two domains of a catalytic monomer. In contrast. ATP, CTP. formycin A 5′-triphosphate and 3, N 6-etheno-ATP bind to an exposed region of this cleft through their β and γ phosphates. Although the β and γ phosphates of 8-bromo-GTP bind to the same region as do the non-brominated nucleotides. 8-bromo-GTP interacts with the protein through all three of its phosphates and its ribose. Ser52, Arg54. Thr55, Arg105, His134. Gln137 and Arg167 are residues of the catalytic chain near density corresponding to phosphate ligands. The interactions of phosphate ligands are consistent with results of nuclear magnetic resonance, kinetics and equilibrium binding studies. Nucleoside triphosphates also bind to the regulatory chain in two modes. ATP and CTP bind in similar conformations to nearly the same site of the allosteric domain. The effector 8-bromo-GTP interacts at a location that does not overlap with the ATP-CTP site. The phosphates are in an extended conformation for all effectors. Furthermore, ATP. 5-bromo-CTP and 8-bromo-GTP bind to the protein in the anti conformation. Interactions of ATP and CTP with the protein are essentially consistent with the proposals put forward by London & Schmidt (1972). We suggest, however, a modification of the London & Schmidt model on the basis of our results with 8-bromo-GTP. In addition, we propose that the allosteric binding sites of nucleoside triphosphates are coupled to each other through the N-terminal segments of monomers of a regulatory dimer.

Crystal and molecular structures of native and CTP-liganded aspartate carbamoyltransferase from Escherichia coli

Atomic models representing the electron density of two crystalline forms of aspartate carbamoyltransferase from Escherichia coli are reported here. The unliganded form ( R32 crystal symmetry) and the CTP-liganded form ( P321 crystal symmetry) have been refined independently at resolutions of 3.0 å and 2.8 Å, respectively, each to a crystallographic R-factor of 27%. The molecular models include at least 95% of the theoretical number of atoms for the aspartate Carbamoyltransferase molecule based on chemical sequence information. We provide details of the refinement process for the two structures, and an evaluation of the accuracy of the molecular models. For the most part, the regulatory and catalytic chains of the unliganded enzyme and the CTP-liganded form are in similar conformations. Large conformational differences in the CTP and native forms exist, however, specifically in the region of CTP binding to the regulatory chain. In addition, a segment of ten amino acid residues, which includes Lys83 and Lys84 of the catalytic chain, is disordered in the CTP-liganded form, in contrast to the native structure, where the same residues have refined well into density. Each catalytic monomer of aspartate carbamoyltransferase is in contact with three catalytic chains and two regulatory monomers. Each regulatory monomer borders on one other regulatory chain and two catalytic chains. The catalytic trimera are in contact in the hexamer; residues important to homotropic effects and catalysis (Tyr165 and Tyr232) are integral parts of the interface. We present a thorough survey of interface regions, cataloging polar interactions between sidechains throughout the molecule. We discuss, in context with the present structures, the chemical modifications and mutations of the enzyme. Highlighted specifically are Cys47, Tyr165 and Tyr232, Lys83, Lys84, Trp209 and Trp279 and Gly128, residues of demonstrated importance to the catalytic of regulatory function or aspartate carbamoyltransferase. The spatial arrangement of “active site” residues argues for a catalytic pocket shared between two monomers within catalytic subunit.

Coupling of homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase

Several types of conditions allow the disconnection of homotropic and heterotropic interactions in Escherichia coli aspartate transcarbamylase. A model that includes a concerted gross conformational change corresponding to the homotropic cooperative interactions between the catalytic sites and local “site by site” effects promoted by the effectors accounts for this disconnection as well as for the other known properties of the enzyme. However, the substrate concentration influences the extent of stimulation and feedback inhibition of the catalytic activity by the effectors. This result is explained by assuming that these effectors promote a “ primary effect”, which is exerted locally “site by site”, and a “ secondary effect”, which is mediated by the substrate. As predicted by the model, relaxed (R) forms of the enzyme show only the primary effect. In addition 2-ThioU-aspartate transcarbamylase, a modified form of the enzyme in which the homotropic cooperative interactions between the catalytic sites are selectively abolished, shows the same heterogeneity in CTP binding sites as normal aspartate transcarbamylase.

Binding of regulatory nucleotides to aspartate transcarbamylase: nuclear magnetic resonance studies of selectively enriched carbon-13 regulatory subunit.

Specifically enriched [gamma-13C]phenylalanine, -tyrosine, and -histidine have been biosynthetically incorporated into aspartate transcarbamylase from Escherichia coli. These nonperturbing NMR probes have been used to characterize the interaction of the regulatory sites on the enzyme with nucleotide effectors. The C gamma carbons of the three tyrosines and four histidines per regulatory chain give narrow, well-resolved resonances, and the signals from the five phenylalanines per chain are partially resolved in the presence of bound inhibitor. Spectral changes in regulatory subunit were monitored as a function of concentration of the inhibitor, CTP, and the activator, ATP. Three histidine residues responded to ATP and CTP in an identical manner while two phenylalanine residues were sensitive to CTP but not ATP binding. The tyrosine resonances were not perturbed by effectors. The chemical shift response of the single observable histidine resonance to bound nucleotides in the reconstituted enzyme was identical with that observed for isolated regulatory subunit. This histidine spectrum was undisturbed by the T to R conformational transition of the enzyme. The results suggest that the regulatory subunit experiences minimal rearrangement of tertiary structure on binding effectors and that at least one phenylalanine and one histidine residue are present in the region of the CTP binding site.

Asymmetry of binding and physical assignments of CTP and ATP sites in aspartate transcarbamoylase.

The allosteric effectors of aspartate transcarbamoylase from Escherichia coli, CTP and ATP, associate with both the regulatory and the catalytic moieties of the enzyme. Studies with isolated, active subunits yield one binding site per regulatory dimer and one per catalytic trimer. Investigations of effector association with hybrid enzymes, containing either the three regulatory dimers or the two catalytic trimers in inactivated forms, indicate that the data obtained with isolated subunits can be used to analyze the binding patterns of these ligands to the native hexamer. Thus, the nonlinear Scatchard plots, characteristic of the binding of CTP and ATP to the native enzyme, can be interpreted in terms of three effector molecules associating with the regulatory subunits, and two binding to the catalytic moiety of the enzyme. Results with native protein in the presence of saturating concentrations of active site ligands support these assignments. The differences between the binding isotherms of CTP and ATP to the enzyme are due to their different affinities to the two types of subunits. The apparent half-of-the-site saturation of the regulatory moiety of aspartate transcarbamoylase supports the concept that this protein has a tendency to exist in an asymmetric state.

Calorimetric analysis of aspartate transcarbamylase from Escherichia coli. Binding of cytosine 5'-triphosphate and adenosine 5'-triphosphate

The binding of CTP and ATP to aspartate transcarbamylase at pH 7.8 and 8.5 at 25 degrees has been investigated by equilibrium dialysis and flow microcalorimetry. The binding isotherms for CTP at both pH 7.8 and 8.5 and ATP AT PH 8.5 can be fit by a model which assumes three tight, three moderately tight, and six weak binding sites. The binding isotherms for ATP at pH 7.8 are best fit by a model which assumes six tight and six weaker sites. Both finite differenceH binding and finite differenceS binding are negative for both nucleotides at both pH values, so that the binding is enthalpy driven. For both nucleotides, finite differenceH is the same for the first two classes of binding sites, implying that the difference in the dissociation constants of these two classes of sites is the result of entropic effects. Direct pH measurements and calorimetric measurements in two buffers with very different heats of ionization (Tris and Hepes) indicate that the binding of both nucleotides is accompanied by the binding of protons. In the pH range 6.7-8.4, the number of moles of protons bound per mole of nucleotide increases as the pH decreases.

Studies on tRNA nucleotidyltransferase from baker's yeast. 1. Purification of the enzyme. Protection against thermal inactivation and inhibition by several substrates.

A rapid purification method for tRNA nucleotidyltransferase from baker's yeast has been worked out. The enzyme, purified 900‐fold with a yield of 20%, is electrophoretically pure.ATP and CTP protect the enzyme against heat inactivation and are competitive inhibitors for each other in the reaction of CCA attachment. Differences in the protection properties, as well as in the kinetic constants, suggest that the ATP and CTP sites on the enzyme are different.The enzyme is also protected by high concentrations of tRNA without the CCA terminus (tRNA‐X), but not by the intact tRNA. Differences between tRNA's with or without the CCA triplet were also found when their inhibitory effect on the attachment of the CCA terminus was studied. It was found that tRNA‐X is a competitive inhibitor in the AMP attachment to tRNA‐XCC, whereas tRNA‐XCCA acts as a non‐competitive inhibitor both for CMP attachment to tRNA‐X and AMP attachment to tRNA‐XCC. Two interpretations for these differences in the behaviour of tRNA with or without the CCA terminus are discussed: (a) either a conformational change of the tRNA when the terminal A is reattached, or (b) the occurrence of a sequential mechanism involving the binding of ATP or CTP to the enzyme prior to the binding of tRNA.

Studies on tRNA nucleotidyltransferase from baker's yeast. 2. Replacement of the terminal CCA sequence in yeast tRNA Phe by several unusual sequences.

Under special conditions, tRNA nucleotidyltransferase is able to add unusual terminal sequences to yeast tRNAPhe whose CCA end has been sequentially removed. By incubating tRNA‐XCCPhe, tRNA‐XCPhe or tRNA‐XPhe with ATP alone, the following tRNAPhe species are obtained: tRNA‐XCCAPhe (normal sequence), tRNA‐XCAPhe and tRNA‐XAPheWhen the degraded tRNAs are incubated with CTP alone, in all cases the sequence tRNA‐XCCCPhe is synthesized, which is able to accept one AMP, thus leading to tRNA‐XCCCAPhe. This compound can be aminoacylated by phenylalanyl‐tRNA synthetase, in contrast to tRNA‐XCAPhe and to tRNA‐XAPhe.

Interaction of Aspartate Transcarbamylase with Regulatory Nucleotides

Abstract Equilibrium dialysis measurements of the binding of the allosteric activator ATP and the allosteric inhibitor CTP to native aspartate transcarbamylase show that ATP is bound at 5.1 to 6 sites with similar affinities, whereas Scatchard plots of CTP binding data display a sharp negative curvature indicating that CTP is bound with markedly heterogeneous affinities. ATP binds more weakly than does CTP, and the binding affinities of both nucleotides are decreased several fold by an increase in temperature from 4° to 24°. The binding affinity of CTP is reduced about 2-fold when the catalytic sites of aspartate transcarbamylase are occupied by a tightly bound transition-state analogue, N-(phosphonacetyl)-l-aspartate (PALA); there is no measurable effect of PALA on the binding of ATP. The heterogeneity in CTP binding affinities is observed under all conditions tested, in a manner consistent with the suggestion that heterogeneity in CTP binding may be due to nonequivalent binding of CTP to sites on dimers of regulatory polypeptide chains in the enzyme. Measurements have been made of the 190 to 320 nm circular dichroism (CD) spectrum of unliganded native aspartate transcarbamylase and of the CD difference spectra arising due to interaction of the enzyme with the substrate analogue PALA, the activator ATP, or the inhibitor CTP, taking these ligands singly or in combination. An estimate of polypeptide configurations in native aspartate transcarbamylase is presented, based on analysis of the 200 to 240 nm CD. Temperature-dependent spectral changes are not detected in the unliganded enzyme but are found in the difference CD at 250 to 320 nm for interaction of ATP or CTP with aspartate transcarbamylase. PALA and CTP added simultaneously to aspartate transcarbamylase have effects on the 200 to 250 nm CD which are not equal to the sum of the effects of PALA or CTP acting separately, reflecting the interdependence in the interaction of these two ligands with aspartate transcarbamylase. PALA, which is an analogue combining features of both of the substrates of aspartate transcarbamylase, produces changes in the 250 to 320 nm CD which are qualitatively and quantitatively similar to the sum of the changes observed by other workers for interaction of aspartate transcarbamylase with one substrate and an analogue of the other substrate.

Conformational Changes in Aspartate Transcarbamylase: II. CIRCULAR DICHROISM EVIDENCE FOR THE INVOLVEMENT OF METAL IONS IN ALLOSTERIC INTERACTIONS

Abstract Replacement of Zn(II) by Cd(II) in aspartate transcarbamylase from Escherichia coli gives rise to an intense circular dichroism difference spectrum with a maximum ellipticity at 259 nm of 44,000 deg cm2 per dmole of Cd(II). This Cotton effect can be assigned to the Cd(II) chromophore and used as a sensitive spectral probe of metal site conformational changes. When circular dichroism difference spectra for carbamyl phosphate and succinate binding to Cd(II)-aspartate transcarbamylase are compared with the corresponding circular dichroism difference spectra for Zn(II)-aspartate transcarbamylase, perturbations of the Cd(II)-chromophore are apparent. Thus, conformational changes at metal ion binding sites in the regulatory subunits occur when substrate or substrate analogue molecules bind at active sites in the catalytic subunits. Conversely, replacement of Zn(II) by Cd(II) slightly alters the kinetic and binding parameters of native aspartate transcarbamylase. Thus, alteration of the metal ion binding site structure alters the conformation of the active site. These observations show that a coupling exists between conformational changes at metal ion sites in regulatory subunits and active sites in catalytic subunits. Consequently, conformational changes at metal ion binding sites may be involved in the allosteric mechanism of homotropic interactions. The binding of the feedback inhibitor, CTP, either to Cd(II)-regulatory subunit or to Cd(II)-aspartate transcarbamylase causes no change in the circular dichroism of the Cd(II)-chromophore. Thus, the metal ion does not seem to be directly involved either in the binding of CTP or in the mechanism of communicating heterotropic interactions. Since the metal ion sites are implicated in homotropic interactions, but not in heterotropic interactions, the allosteric mechanisms of these two effects must involve different sets of conformational changes. To assess the linkage between conformational changes at different loci in aspartate transcarbamylase, circular dichroism changes at different wave lengths were monitored during ligand titrations. For Cd(II)-aspartate transcarbamylase, the apparent carbamyl phosphate concentration required for half-maximal change is 0.32 mm at 280 nm (tyrosyl chromophore) but 0.10 mm at 256 nm (Cd(II)-chromophore). The succinate concentration required for half-maximal change is 0.20 mm at 256 nm (Cd(II)-chromophore) but 0.40 mm at 290 nm (tryptophanyl chromophore). These results demonstrate the existence of multiple conformational equilibria for the binding of these substrate ligands and suggest that allosteric interactions of aspartate transcarbamylase cannot adequately be described by a two-state model.

Reactions at the 3′ Terminus of Transfer Ribonucleic Acid: III. CATALYTIC PROPERTIES OF TWO PURIFIED RABBIT LIVER TRANSFER RIBONUCLEIC ACID NUCLEOTIDYLTRANSFERASES

Abstract The catalytic properties of two purified rabbit liver tRNA nucleotidyltransferases were examined and compared. Both enzymes incorporated AMP, CMP, and UMP into tRNA, but were inactive with GTP or dATP. The pH optima for the various nucleotides and for both enzymes were similar and were in the range of pH 9 to 10. Differences were observed in the response of the two enzymes to the assay temperature, such that the activation energies for phosphocellulose Peak II were about 50% higher than for Peak I. The apparent Km values for nucleoside triphosphates were essentially identical for both enzymes. For ATP with tRNApCpC as cosubstrate the Km values were 3 mm and 2 mm for enzymes I and II, respectively; using tRNApC as cosubstrate the apparent Km with Peak II was 2 mm. In the case of CTP, biphasic double reciprocal plots were found which suggested the existence of two CTP binding sites. The data for each enzyme could be fit to curves generated from Km values of 0.004 mm and 0.4 mm and appropriate Vmax values. Similar Km values for CTP were obtained with tRNApC or tRNApX as cosubstrate although the Vmax values differed. UTP, which acted as an analogue of CTP, gave apparent Km values of 0.06 mm and 0.05 mm with enzymes I and II using tRNApC as cosubstrate. Both tRNA nucleotidyltransferases could utilize Mg++, Mn++, and Co++ to satisfy the requirement for divalent cation, but the latter two ions were less effective than Mg++. The rate of nucleotide incorporation into tRNAs with defined termini, as well as into other nucleic acids, was examined. tRNApCpCpA, rRNA, calf thymus DNA, or poly(C) were inactive as acceptors for either AMP, CMP, or UMP. tRNApCpC was a substrate for AMP incorporation, but was inactive with CTP or UTP. tRNApC and tRNApX were substrates for both CMP and UMP incorporation, but displayed diminishing activity in the presence of ATP. The apparent Km values for the various tRNA substrates were in the range of 4 to 30 µm, and were increased by the incorrect triphosphate. Liver tRNA nucleotidyltransferase showed no species specificity for the source of tRNA, being equally active with material from rabbit liver, yeast, and Escherichia coli. The extents of AMP, CMP, and UMP incorporation into tRNApC catalyzed by each enzyme were essentially the same suggesting that both enzymes probably acted on the same tRNA population. Another difference between the two tRNA nucleotidyltransferases was their response to a variety of salts. AMP incorporation catalyzed by either enzyme was inhibited by all salts examined. On the other hand, CMP incorporation catalyzed by Peak I was relatively little affected by monovalent salts at ionic strengths below 0.25, and that catalyzed by Peak II was greatly stimulated. These results point out that although many of the properties of the two enzymes are quite similar, significant differences do exist. A possible model for tRNA nucleotidyltransferase action is also presented.