Reversal of the effect of the allosteric ligands of dCMP-aminohydrolase and stabilization of the enzyme in the T form

The 5-mercury derivative of dCMP is a substrate of deoxycytidylate aminohydrolase in the presence of mercaptoethanol. With this substrate a reversal of the effect of the allosteric ligands of the enzyme is observed. dCTP, which is an allosteric activator for aminohydrolysis of dCMP, becomes an inhibitor for the mercury substrate, whilst dTTP, an allosteric inhibitor for dCMP, becomes an activator for the mercury substrate.This observation has been interpreted by assuming that dCMP-Hg-S-CH₂-CH₂-OH is a substrate of the T form of the enzyme. By reacting dCMP-aminohydrolase in the T form (in the presence of dTTP) with glutaraldehyde, an enzyme has been isolated that is no longer active with dCMP, while it is fully active with the mercurated analog. Gel electrophoresis demonstrated that glutaraldehyde does not produce intermolecular crosslinks, but fixes 95% of the enzyme in a stable hexameric form by intramolecular crosslinks. The data are explained by assuming that glutaraldehyde stabilizes the enzyme in the T conformation.     

The First Crystal Structure of a dTTP-bound Deoxycytidylate Deaminase Validates and Details the Allosteric-Inhibitor Binding Site

Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH₂ in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed.     

Deoxycytidine triphosphate deaminase of Salmonella typhimurium. Purification and characterization.

Deoxycytidine triphosphate deaminase (EC 3.5.4., dCTP aminohydrolase) of Salmonella typhimurium LT2 has been pruified 500-fold. The reaction requires the presence of Mg-2plus, Mn-2plus, Ca-2lus, or Co-2plus. Kinetics of the reaction with varying Mg-2plus concentrations, keeping the concentration of dCTP constant, suggests that the true substrate of the reaction is MgdCTP. The dependence of the rate of reaction on dCTP concentration in the presnece of 5-fold excess of Mg-2plus is sigmoid, with a Hill coefficient of 1.7. The enzyme is specifically inhibited by dTTP and dUTP. In the presence of increasing dTTP concentrations the sigmoidicity of the substrate saturation curves increases. With 0.2 and 0.4 mM dTTP the Hill coefficients are 2.6 and 3.0, respectively. Despite several attempts no dissociation of the substrate site and the inhibitor site of the enzyme was achieved.     

Deoxycytidylate deaminase activity in non-stimulated and phytohemagglutinin-stimulated human lymphocytes,and in leukemic cells

Deoxycytidylate deaminase isolated from normal human lymphocytes and from mononuclear leucocytes from patients with acute lymphoblastic leukemia, chronic lymphocytic leukemia and acute monocytic leukemia has been characterized in regard to the substrate, dAMP and the allosteric regulators dCTP and dTTP. The enzymes exhibited sigmoidal initial velocity versus dCMP concentration whereas in the presence of the activator, dCTP, Michaelis-Menten kinetics were obtained.At saturating substrate concentrations dTTP acted as an allosteric inhibitor of the enzyme isolated from non-stimulated as well as from stimulated lymphocytes. However, the enzymes isolated from the leukemic cells had lost the allosteric regulation by dTTP.At low substrate concentrations the competitive inhibitor, dAMP, activated all the enzymes. This activation was abolished in the presence of dCTP which indicates that dAMP might be involved in the regulation of dCMP deaminase activity and thus influence the dCTP and dTTP pools under physiological conditions.Abbreviations dCMP deaminase deoxycytidylate deaminase - PHA Phytohemagglutinin - ALL acute lymphoblastic leukemia - CLL chronic lymphocytic leukemia - AMOL acute monocytic leukemia - WBC white blood cells     

Regulation of dCTP deaminase from Escherichia coli by nonallosteric dTTP binding to an inactive form of the enzyme

The trimeric dCTP deaminase produces dUTP that is hydrolysed to dUMP by the structurally closely related dUTPase. This pathway provides 70-80% of the total dUMP as a precursor for dTTP. Accordingly, dCTP deaminase is regulated by dTTP, which increases the substrate concentration for half-maximal activity and the cooperativity of dCTP saturation. Likewise, increasing concentrations of dCTP increase the cooperativity of dTTP inhibition. Previous structural studies showed that the complexes of inactive mutant protein, E138A, with dUTP or dCTP bound, and wild-type enzyme with dUTP bound were all highly similar and characterized by having an ordered C-terminal. When comparing with a new structure in which dTTP is bound to the active site of E138A, the region between Val120 and His125 was found to be in a new conformation. This and the previous conformation were mutually exclusive within the trimer. Also, the dCTP complex of the inactive H121A was found to have residues 120-125 in this new conformation, indicating that it renders the enzyme inactive. The C-terminal fold was found to be disordered for both new complexes. We suggest that the cooperative kinetics are imposed by a dTTP-dependent lag of product formation observed in presteady-state kinetics. This lag may be derived from a slow equilibration between an inactive and an active conformation of dCTP deaminase represented by the dTTP complex and the dUTP/dCTP complex, respectively. The dCTP deaminase then resembles a simple concerted system subjected to effector binding, but without the use of an allosteric site.     

Mammalian thymidine kinase 2. Direct photoaffinity labeling with [32P]dTTP of the enzyme from spleen, liver, heart and brain.

Thymidine kinase 2 (TK2), also called mitochondrial thymidine kinase, is a pyrimidine deoxyribonucleoside kinase expressed in all cells and tissues. It was recently purified to apparent homogeneity from human leukemic spleen and the active enzyme was shown to be a monomer of a 29-kDa polypeptide. The enzyme is feedback-inhibited by both end products, dCTP and dTTP. Here we show that TK2 purified from several different sources, including purified beef heart mitochondria, could be directly photoaffinity labeled with radioactive dTTP (approximately 18% of all TK2 molecules were cross-linked to dTTP after 20 min of ultraviolet irradiation) or to a lower extent with dCTP. Photo-incorporation was inhibited by the presence of the other effector but also the phosphate donor ATP blocked photolabeling, with dTTP. Addition of nucleoside substrates gave only a marginal inhibition of photo-incorporation. There were no detectable difference in the molecular size of photolabeled TK2 isolated from human spleen, brain or placenta, monkey liver, beef heart and beef heart mitochondria. Nor was there any significant differences in the enzyme kinetic properties of these enzymes. Cleavage of labeled TK2 with cyanogen bromide showed that dTTP was incorporated into a single 3-kDa peptide. TK2 was the only pyrimidine deoxynucleoside kinase expressed in liver, heart and brain. A detailed characterization of the subunit structure and substrate specificity of this enzyme is of importance for the design of new antiviral and cytostatic therapies based on nucleoside analogs.     

Hill coefficient ratios give binding ratios of allosteric enzyme effectors; inhibition, activation, and squatting in deoxycytidylate aminohydrolase (EC 3.5.4.12)

The ratio of the steady-state kinetic Hill coefficients of two different effectors equals (under some rather weak general assumptions) the ratio in which the effectors displace each other from an enzyme. This principle can make implications of experimental allosteric enzyme kinetic data immediately apparent. We can use it to find that one molecule of the allosteric inhibitor of dCMP aminohydrolase, at moderately high effector concentrations, displaces one molecule of substrate, or one molecule of activator, whereas at very high concentrations, one molecule of inhibitor displaces two of substrate. Further use of the principle suggests that substrate, at high concentrations, binds binds to activator sites. However, ratios of substrate, activator, and inhibitor Hill coefficients are incompatible with a simple model of activation in which substrate and activator are bound to the same conformation.     

Structural basis for recognition and catalysis by the bifunctional dCTP deaminase and dUTPase from Methanococcus jannaschii

Potentially mutagenic uracil-containing nucleotide intermediates are generated by deamination of dCTP, either spontaneously or enzymatically as the first step in the conversion of dCTP to dTTP. dUTPases convert dUTP to dUMP, thus avoiding the misincorporation of dUTP into DNA and creating the substrate for the next enzyme in the dTTP synthetic pathway, thymidylate synthase. Although dCTP deaminase and dUTPase activities are usually found in separate but homologous enzymes, the hyperthermophile Methanococcus jannaschii has an enzyme, DCD-DUT, that harbors both dCTP deaminase and dUTP pyrophosphatase activities. DCD-DUT has highest activity on dCTP, followed by dUTP, and dTTP inhibits both the deaminase and pyrophosphatase activities. To help clarify structure-function relationships for DCD-DUT, we have determined the crystal structure of the wild-type DCD-DUT protein in its apo form to 1.42A and structures of DCD-DUT in complex with dCTP and dUTP to resolutions of 1.77A and 2.10A, respectively. To gain insights into substrate interactions, we complemented analyses of the experimentally defined weak density for nucleotides with automated docking experiments using dCTP, dUTP, and dTTP. DCD-DUT is a hexamer, unlike the homologous dUTPases, and its subunits contain several insertions and substitutions different from the dUTPase beta barrel core that likely contribute to dCTP specificity and deamination. These first structures of a dCTP deaminase reveal a probable role for an unstructured C-terminal region different from that of the dUTPases and possible mechanisms for both bifunctional enzyme activity and feedback inhibition by dTTP.     

Ribonucleotide reductase from Escherichia coli. Identification of allosteric effector sites by chromatography on immobilized effectors.

Ribonucleotide reductase is responsible for the production of deoxyribonucleotides by catalyzing the reduction of ribonucleoside diphosphates. The enzyme is allosterically regulated in a complex way by the nucleoside triphosphates, ATP, dTTP, dGTP, dCTP, and dATP. Ribonucleotide reductase consists of two nonidentical subunits, proteins B1 and B2. Both substrates and allosteric effectors bind exclusively to B1. Binding of protein B1 to dTTP or dATP covalently coupled to Sepharose and elution with concentration gradients of the different nucleoside triphosphate effectors gave information about (1) the arrangement of the effector binding sites on protein B1 and (2) the affinity of the effectors for these sites. Protein B1 thus has two classes of effector binding sites. One class binds all effectors, as demonstrated by elution of the protein from dTTP-Sepharose with dATP, dGTP, ATP, or dCTP. The second class binds only dATP or ATP, since dATP and ATP were the only nucleotides which eluted protein B1 from dATP-Sepharose. These results confirm earlier data obtained by dialysis binding experiments. The eluting concentrations obtained for the different nucleoside triphosphates in experiments with dTTP-Sepharose could be used to calculate unknown dissociation constants for protein B1 -effector binary complexes. This was possible, since a plot of the eluting concentrations vs. known dissociation constants was linear.     

Ribonucleotide reductase activity and deoxyribonucleoside triphosphate metabolism during the cell cycle of S49 wild-type and mutant mouse T-lymphoma cells

We investigated deoxyribonucleoside triphosphate metabolism in S49 mouse T-lymphoma cells synchronized in different phases of the cell cycle. S49 wild-type cultures enriched for G1 phase cells by exposure to dibutyryl cyclic AMP (Bt2cAMP) for 24 h had lower dCTP and dTTP pools but equivalent or increased pools of dATP and dGTP when compared with exponentially growing wild-type cells. Release from Bt2cAMP arrest resulted in a maximum enrichment of S phase occurring 24 h after removal of the Bt2cAMP, and was accompanied by an increase in dCTP and dTTP levels that persisted in colcemid-treated (G2/M phase enriched) cultures. Ribonucleotide reductase activity in permeabilized cells was low in G1 arrested cells, increased in S phase enriched cultures and further increased in G2/M enriched cultures. In cell lines heterozygous for mutations in the allosteric binding sites on the M1 subunit of ribonucleotide reductase, the deoxyribonucleotide pools in S phase enriched cultures were larger than in wild-type S49 cells, suggesting that feedback inhibition of ribonucleotide reductase is an important mechanism limiting the size of deoxyribonucleoside triphosphate pools. The M1 and M2 subunits of ribonucleotide reductase from wild-type S49 cells were identified on two-dimensional polyacrylamide gels, but showed no significant change in intensity during the cell cycle. These data are consistent with allosteric inhibition of ribonucleotide reductase during the G1 phase of the cycle and release of this inhibition during S phase. They suggest that the increase in ribonucleotide reductase activity observed in permeabilized S phase-enriched cultures may not be the result of increased synthesis of either the M1 or M2 subunit of the enzyme.     

Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase iota

O(6)-methylguanine (O(6)-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was determined for nucleoside triphosphate incorporation opposite O(6)-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O(6)-methylG. Misincorporation of dTTP opposite O(6)-methylG occurred with ～6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O(6)-methylG as the template base and incoming dCTP or dTTP were solved and showed that O(6)-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O(6)-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O(6)-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O(6) atoms of O(6)-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O(6)-methylG by human pol ι, in contrast to the mispairing modes observed previously for O(6)-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.     

Mutational analysis of the nucleotide binding site of Escherichia coli dCTP deaminase

In Escherichia coli and Salmonella typhimurium about 80% of the dUMP used for dTMP synthesis is derived from deamination of dCTP. The dCTP deaminase produces dUTP that subsequently is hydrolyzed by dUTPase to dUMP and diphosphate. The dCTP deaminase is regulated by dTTP that inhibits the enzyme by binding to the active site and induces an inactive conformation of the trimeric enzyme. We have analyzed the role of residues previously suggested to play a role in catalysis. The mutant enzymes R115Q, S111C, S111T and E138D were all purified and analyzed for activity. Only S111T and E138D displayed detectable activity with a 30- and 140-fold reduction in k_cat, respectively. Furthermore, S111T and E138D both showed altered dTTP inhibition compared to wild-type enzyme. S111T was almost insensitive to the presence of dTTP. With the E138D enzyme the dTTP dependent increase in cooperativity of dCTP saturation was absent, although the dTTP inhibition itself was still cooperative. Modeling of the active site of the S111T enzyme indicated that this enzyme is restricted in forming the inactive dTTP binding conformer due to steric hindrance by the additional methyl group in threonine. The crystal structure of E138D in complex with dUTP showed a hydrogen bonding network in the active site similar to wild-type enzyme. However, changes in the hydrogen bond lengths between the carboxylate and a catalytic water molecule as well as a slightly different orientation of the pyrimidine ring of the bound nucleotide may provide an explanation for the reduced activity.     

AMP deaminase from baker's yeast. Kinetic and molecular properties.

The kinetic and molecular properties of AMP deaminase [AMP aminohydrolase, EC 3.5.4.6] purified from baker's yeast (saccharomyces cerevisiae) were investigated. The enzyme was activated by ATP and dATP, but inhibited by Pi and GTP in an allosteric manner. Alkali metal ions and alkaline earth metal ions activated the enzyme to various extent. Kinetic negative cooperativity was observed in the binding of nucleoside triphosphates. Kinetic analysis showed that the number of interaction sites for AMP (substrate) and Pi (inhibitor) is two each per enzyme molecule. The molecular weight of the native enzyme was estimated to be 360,000 by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme gave a single polypeptide band with a molecular weight of 83,000, suggesting that the native enzyme has a tetrameric structure. Baker's yeast AMP deaminase was concluded to consist of two "promoter" units which each consist of two polypeptide chains with identical molecular weight.     

Freezing of dCMP aminohydrolase in the activated conformation by glutaraldehyde.

dCMP aminohydrolase, which is an allosteric enzyme, was reacted with glutaraldehyde in the presence of the allosteric activator deoxycytidine-5′-triphosphate and of the competitive inhibitor deoxyadenosine-5′-monophosphate. The isolated modified enzyme is no longer sensitive to the effect of the allosteric ligands and shows kinetics typical of the activated enzyme. Gel electrophoresis demonstrated that glutaraldehyde, under our experimental conditions, does not produce intermolecular cross-links but fixes 80% of the enzyme in a stable hexameric form by intramolecular cross-links.The kinetic and molecular data are explained assuming that glutaraldehyde freezes the enzyme in the activated conformation.     

Quantitation of cellular deoxynucleoside triphosphates

Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP.     

Biosynthesis of deoxynucleoside triphosphates,dCTP and dTTP: reaction mechanism and kinetics

The enzyme reaction mechanism and kinetics for biosyntheses of deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP) from the corresponding deoxycytidine diphosphate (dCDP) and deoxythymidine diphosphate (dTDP) catalyzed by pyruvate kinase were studied. The kinetic model for the two synthetic reactions was found to follow the Bi–Bi random rapid equilibrium mechanism similar to that of the biosynthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP). Kinetic constants involved in the reactions including the maximum reaction velocity, the Michaelis–Menten constants, and the inhibition constants for dCTP and dTTP biosyntheses were experimentally determined. This enzyme reaction requires Mg²⁺ ion and the optimal Mg²⁺ concentration was also determined. The experimental results showed a good agreement with the simulation results obtained from the kinetic model developed. The kinetics of the four biosynthetic reactions for deoxynucleoside triphosphates (dNTP) including dATP, dGTP, dCTP, and dTTP from the corresponding deoxynucleoside diphosphates (dNDP) including dADP, dGDP, dCDP, and dTDP were analyzed. The results suggest that the binding kinetics of phosphoenolpyruvate (PEP) and pyruvate are similar for all four biosynthetic reactions. The affinity of the dNDP substrates to enzyme is of the same order of magnitude as the corresponding dNTP as inhibitors. The order of reactivity and substrate specificity for dNDP is dADP > dGDP > dCDP > dTDP in the pyruvate kinase (PK) reactions. The results obtained from this study can be applied to bioreactor design and production of dCTP and dTTP for biosynthesis of DNA at a significantly lower cost compared to the currently available chemical method.     

T4 phage ribonucleotide reductase. Allosteric regulation in vivo by thymidine triphosphate

Based upon analyses of purified enzyme preparations, T4 bacteriophage-coded ribonucleotide reductase is considered to be relatively insensitive to control by allosteric inhibition. However, two factors suggest that CDP reduction to dCDP is feedback-controlled by dTTP in infected cells. First, the pool of 5-hydroxymethyldeoxycytidine triphosphate, which expands manyfold upon infection by a dCMP deaminase-deficient T4 mutant, shrinks to near-normal levels as a consequence of dTTP accumulation, and ribonucleotide reductase is the only apparent control point. Second, analysis of mutagenesis by 5-bromodeoxyuridine suggests that most induced mutations result from localized pool depletion of 5-hydroxymethyl-dCTP at replication sites, as if 5-bromo-dUTP were behaving like dTTP in inhibiting the CDP reductase activity of the phage enzyme. We found that CDP reductase activity in crude extracts of T4 phage-infected bacteria is sensitive to inhibition by either dTTP or 5-bromo-dUTP, at concentrations as low as 0.01 mM. However, in partially purified enzyme preparations that sensitivity is lost. Although we don't know the basis for this loss of feedback sensitivity, the results suggest that kinetic properties of enzymes in intact cells are determined by the cellular milieu in ways not apparent from analysis of purified enzymes.     

Mechanisms of Allosteric Activation and Inhibition of the Deoxyribonucleoside Triphosphate Triphosphohydrolase from Enterococcus faecalis

EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity.     

Structure of the large subunit of class Ib ribonucleotide reductase from Salmonella typhimurium and its complexes with allosteric effectors

The three-dimensional structure of the large subunit of the first member of a class Ib ribonucleotide reductase, R1E of Salmonella typhimurium, has been determined in its native form and together with three allosteric effectors. The enzyme contains the characteristic ten-stranded alpha/beta-barrel with catalytic residues at a finger loop in its center and with redox-active cysteine residues at two adjacent barrel strands. Structures where the redox-active cysteine residues are in reduced thiol form and in oxidized disulfide form have been determined revealing local structural changes. The R1E enzyme differs from the class Ia enzyme, Escherichia coli R1, by not having an overall allosteric regulation. This is explained from the structure by differences in the N-terminal domain, which is about 50 residues shorter and lacks the overall allosteric binding site. R1E has an allosteric substrate specificity regulation site and the binding site for the nucleotide effectors is located at the dimer interface similarly as for the class Ia enzymes. We have determined the structures of R1E in the absence of effectors and with dTTP, dATP and dCTP bound. The low affinity for ATP at the specificity site is explained by a tyrosine, which hinders nucleotides containing a 2'-OH group to bind.     

Deoxyribonucleotide pools in mouse-fibroblast cell lines with altered ribonucleotide reductase.

Mutant cells lines of 3T6 mouse fibroblasts, resistant to thymidine and deoxyadenosine, have an altered allosteric regulation of the enzyme ribonucleotide reductase (Meuth, M. and Green, H., Cell, 3, 367, 1974). Compared to 3T6, these lines contain larger pools of deoxynucleoside triphosphates, in particular deoxycytidine triphosphate, but show a normal rate of DNA synthesis. Addition of thymidine or deoxyadenosine to 3T6 cells results in large accumulations of the corresponding triphosphates and a dramatic decrease in the dCTP pool, concomitant with inhibition of DNA synthesis. Addition of thymidine to the mutant cell lines also leads to an increase in the dTTP pool but does not result in a depletion of dCTP or inhibition of DNA synthesis. Addition of deoxyadenosine only leads to a small increase of the dATP pool. In general the change in the allosteric regulation of bibonucleotide reductase is reflected in the deoxynucleotide pools.