Requirement of protein synthesis for the induction of ribonucleoside diphosphate reductase mRNA in Escherichia coli

Summary An RNA-DNA hybridization assay was used to quantitate the ribonucleoside diphosphate reductase mRNA synthesis (nrd mRNA) to show that gene expression was dependent on protein synthesis. The increased nrd mRNA synthesis induced by inhibition of DNA synthesis was eliminated by simultaneous inhibition of protein synthesis. It was further found that protein synthesis is required not only initially but continuously during DNA inhibition for increased expression of nrd mRNA synthesis.     

Coordinate control of the synthesis of ribonucleoside diphosphate reductase components in Escherichia coli.

Ribonucleoside diphosphate reductase subunits B1 and B2 and ether-permeabilized cell activities of Escherichia coli increase in parallel during thymine deprivation. Thioredoxin and thioredoxin reductase activities are not affected by thymine deprivation.     

Mechanism of ribonucleoside diphosphate reductase from Escherichia coli. Evidence for 3'-C--H bond cleavage

Incubation of the pyrimidine [3'-3H]UDP with ribonucleotide reductase resulted in an isotope effect on the conversion to dUDP which varied as a function of pH and allosteric effectors (pH, kH/kT, effector): 6.6, 4.7, ATP; 7.6, 3.3, ATP; 7.6, 2.6, dATP; 7.6, 2.0, TTP; 8.4, 2.8, ATP. During this reaction 3H2O was also released. The lower the pH of the reaction, the larger the isotope effect, and the smaller the amount of 3H2O produced. At 50% conversion of UDP to dUDP and at pH 7.6, approximately 0.5% of total 3H present in solution was volatilized, while at pH 8.4, approximately 0.9% was volatilized. Similar experiments in which the purine [3'-3H]ADP was incubated with ribonucleotide reductase also resulted in an isotope effect on its conversion to dATP which varied as a function of pH (pH, kH/kT with dGTP as an effector); 6.6, 1.9; 7.6, 1.7; 8.6, 1.4. Furthermore, 3H2O was also released as a function of the extent of the reaction. At 50% turnover and pH 7.6, approximately 0.6% of 3H2O was volatilized, while at pH 8.6 approximately 1.25% was released. Two control experiments in which either the B1 subunit of ribonucleotide reductase was inactivated with 2'-chloro-2'-deoxyuridine 5'-diphosphate or the B2 subunit of ribonucleotide reductase was inactivated with 2'-azido-2'-deoxyuridine 5'-diphosphate and then the enzyme incubated with [3'-3H]ADP or [3'-3H]UDP indicated that in neither case was 3H released. Both B1 and B2 subunits are required for cleavage of the 3'-C--H bond. Incubation of [3'-3H]dADP or [3'-3H]dUDP with ribonucleotide reductase produced no measurable release of 3H. These data clearly indicate that conversion of a purine or pyrimidine diphosphate to a deoxynucleotide diphosphate by Escherichia coli ribonucleotide reductase requires cleavage of the 3'-C--H bond of the substrate. The fate of the 3'-H of the substrate was also determined. Incubation of [3'-2H]UDP with ribonucleotide reductase resulted in the production of [3'-2H]dUDP.     

Cell cycle regulation of ribonucleoside diphosphate reductase activity in permeable mouse L cells and in extracts

Ribonucleoside diphosphate reductase (EC1.17.4.1) was previously characterized in exponentially growing mouse L cells selectively permeabilized to small molecules by treatment with dextran sulfate (Kucera and Paulus, 1982b). This characterization has now been extended to cells in specific phases of the cell cycle and in transition between cell cycle phases, with activity studied both in situ (permeabilized cells) and in cell extracts. Cells at various stages in the cell cycle were obtained by unit-gravity sedimentation employing a commercially available reorienting chamber device, by G1 arrest induced by isoleucine limitation, and by metaphase arrest induced by Colcemid. G1 cells from both cycling and noncycling populations had negligible levels of ribonucleotide reductase activity as measured by CDP reduction both in situ and in extracts. When G1 arrested cells were allowed to progress to S phase, ribonucleotide reductase activity increased in parallel with [3H]thymidine incorporation into DNA. Ribonucleotide reductase activity in extracts increased at a somewhat greater rate than in situ activity. S phase ribonucleotide reductase activity measured in situ resembled the previously characterized activity in exponentially growing cells with respect to an absolute dependence on ATP or its analogs as positive allosteric effector, sensitivity to the negative allosteric effector dATP, and low susceptibility to stimulation by NADPH, dithiothreitol, and FeCl3. Disruption of permeabilized cells caused reductase activity to become highly dependent on the presence of both dithiothreitol and FeCl3. As synchronized cultures progressed from S into G2/M phase, no significant change in ribonucleotide reductase activity was seen. On the other hand, when cells that had been arrested in metaphase by Colcemid were allowed to resume cell cycle traversal by removing the drug, in situ ribonucleotide reductase activity decreased by 75% within 2.5 h. This decrease seemed to be a late mitotic event, since it was not correlated with the percentage of cells entering G1 phase. The cause of a subsequent slight increase of in situ ribonucleotide reductase activity is not clear. Parallel measurements of ribonucleotide reductase activity in cell extracts indicated also an initial decline accompanied by increasing dependence on added dithiols and FeCl3, followed by complete activity loss. Our results suggest a cell cycle pattern of ribonucleotide reductase activity that involves negligible levels in G1 phase, a progressive increase of activity upon entry into S phase paralleling overall DNA synthesis, continued retention of significant ribonucleotide reductase activity well into the metaphase period of mitosis, and a very rapid decline in activity during the later phases of mitosis. The periods of increase and decrease of ribonucleotide reductase activity were accompanied by modulation of the properties of the enzyme as indicated by differential changes in enzyme activity measured in situ and in extracts.     

A pre-steady state analysis of ligand binding to human glucokinase: evidence for a preexisting equilibrium

Cooperativity with glucose is a key feature of human glucokinase (GK), allowing its crucial role as a glucose sensor in hepatic and pancreatic cells. We studied the changes in enzyme intrinsic tryptophan fluorescence induced by binding of different ligands to this monomeric enzyme using stopped-flow and equilibrium binding methods. Glucose binding data under pre-steady state conditions suggest that the free enzyme in solution is in a preexisting equilibrium between at least two conformers (super-open and open) which differ in their affinity for glucose (Kd* = 0.17 +/- 0.02 mM and Kd = 73 +/- 18 mM). Increasing the glucose concentration changes the ratio of the two conformers, thus yielding an apparent Kd of 3 mM (different from a Km of 7-10 mM). The rates of conformational transitions of free and GK complexed with sugar are slow and during catalysis are most likely affected by ATP binding, phosphate transfer, and product release steps to allow the kcat to be 60 s-1. The ATP analogue PNP-AMP binds to free GK (super-open) and GK-glucose (open) complexes with comparable affinities (Kd = 0.23 +/- 0.02 and 0.19 +/- 0.08 mM, respectively). However, cooperativity with PNP-AMP observed under equilibrium binding conditions in the presence of glucose (Hill slope of 1.6) is indicative of further complex tightening to the closed conformation. Another physiological modulator (inhibitor), palmitoyl-CoA, binds to GK with similar characteristics, suggesting that conformational changes induced upon ligand binding are not restricted by an active site ligand. In conclusion, our data support control of GK activity and Km through the ratio of distinct conformers (super-open, open, and closed) through either substrate or other ligand binding and/or dissociation.     

Formation of the cystine between cysteine 225 and cysteine 462 from ribonucleoside diphosphate reductase is kinetically competent

Participation of the formation of the cystine between cysteine 225 and cysteine 462 in the R1 protein to the enzymatic mechanism of aerobic ribonucleoside diphosphate reductase from Escherichia coli has been examined by use of rapid quenching and site-directed immunochemistry. Prereduced ribonucleotide reductase in the presence of ATP was mixed with CDP in a quench flow apparatus. The reaction was terminated with a solution of acetic acid and N-ethylmaleimide. The protein was precipitated and digested with chymotrypsin and the proteinase from Staphylococcus aureus strain V8 in the presence of N-ethylmaleimide to yield the peptide SS[S-(N-ethylsuccinimid-2-yl)cysteinyl]VLIE containing cysteine 225 and the mixed disulfide between the peptide SSCVLIE and the peptide IALCTL containing cysteine 462. These two peptides were retrieved together from the digest by immunoadsorption. The affinity-purified peptides were modified at their amino termini with the fluorescent reagent 6-aminoquinolyl-N-hydroxysuccimidyl carbamate and submitted to high-pressure liquid chromatography. The areas of the respective peaks of fluorescence corresponding to the S-(N-ethylsuccimidyl) peptide, and the mixed disulfide were used to determine the percentage of the cystine that had formed during each interval. The rate constant for the formation of the cystine following the association of free, fully reduced ribonucleotide reductase with the reactant CDP was 8 s(-)(1). Because only 50% of the active sites participated in this pre-steady-state reaction, the maximum steady-state rate consistent with the involvement of this cystine in the enzymatic reaction would be 4 s(-1). Since the turnover number of the enzyme under the same conditions in a steady state assay was only 1 s(-)(1), the formation of the cystine between these two cysteines is kinetically competent.     

Products of the inactivation of ribonucleoside diphosphate reductase from Escherichia coli with 2'-azido-2'-deoxyuridine 5'-diphosphate

Ribonucleoside diphosphate reductase (RDPR) from Escherichia coli was completely inactivated by 1 equiv of the mechanism-based inhibitor 2'-azido-2'-deoxyuridine 5'-diphosphate (N3UDP). Incubation of RDPR with [3'-3H]N3UDP resulted in 0.2 mol of 3H released to solvent per mole of enzyme inactivated, indicating that cleavage of the 3' carbon-hydrogen bond occurred in the reaction. Incubation of RDPR with [beta-32P]N3UDP resulted in stoichiometric production of inorganic pyrophosphate. One equivalent of uracil was eliminated from N3UDP, but no azide release was detected. Analysis of the reaction of RDPR with [15N3]N3UDP by mass spectrometry revealed that the azide moiety was converted to 0.9 mol of nitrogen gas per mole of enzyme inactivated. The tyrosyl radical of the B2 subunit was destroyed during the inactivation by N3UDP as reported previously [Sj?berg, B.-M., Gr?slund, A., & Eckstein, F. (1983) J. Biol. Chem. 258, 8060-8067], while the specific activity of the B1 subunit was reduced by half. Incubation of [5'-3H]N3UDP with RDPR resulted in stoichiometric covalent radiolabeling of the enzyme. Separation of the enzyme's subunits by chromatofocusing revealed that the modification was specific for the B1 subunit.     

Kinetics of ternary complex formation between dihydrofolate reductase, coenzyme, and inhibitors

The kinetics of ligand binding to dihydrofolate reductase from Lactobacillus casei (MTX/R) to form the ternary enzyme-inhibitor-coenzyme complex have been investigated by the stopped-flow fluorescence technique. The fluorescence changes observed when coenzymes or inhibitors bind to the binary complex of the enzyme with the complementary ligand occur in a single fast phase. Under pseudo-first-order conditions the reaction traces could be fitted with precision to a single-exponential decay, and apparent bimolecular rate constants in the range 2 x 10(6) to 3 x 10(7) M-1s-1 have been measured assuming a bimolecular-unimolecular model. The kinetic constants obtained suggest that prior binding of an inhibitor to the enzyme may, to a minor extent, interfere with coenzyme binding but the rates of inhibitor binding seem to be unaffected by the presence of a bound coenzyme. Dissociation rate constants appear to be less than 1 s-1 which suggests that both coenzymes and inhibitors are tightly bound in the ternary complex. An investigation of the effects of pH on the kinetics of ternary complex formation indicated the involvement of ionizable groups in ligand binding, but this shows some ligand dependence. The rates of ligand bindings to form the ternary complex are fairly high, but it is unlikely that these associations are diffusion controlled because their measured activation energies of 7.8-14.5 kcal mol-1 are higher than expected from reactions whose rates are limited by diffusion in aqeous solution.     

Regulation of the synthesis of ribonucleoside diphosphate reductase in Escherichia coli: specific activity of the enzyme in relationship to perturbations of DNA replication.

Ribonucleoside diphosphate reductase (RDP reductase) activity was found to greatly increase after a shift to the nonpermissive temperature in Escherichia coli mutants temperature sensitive for DNA elongation (dnaE dnaG dnaZ lig) or DNA initiation (dnaA dnaC dnaI). However, the kinetics of increase in RDP reductase after a shift to nonpermissive conditions were significantly different in initiation-defective mutants compared with elongation-defective mutants. In strains without defects in DNA metabolism, the specific activity of RDP reductase was found to increase with increasing growth rate. Nutritional shifts to faster growth conditions caused cells to transiently overproduce RDP reductase before adjusting to the new steady-state conditions.     

Synthesis of 3'-C-methyladenosine and 3'-C-methyluridine diphosphates and their interaction with the ribonucleoside diphosphate reductase from Corynebacterium nephridii.

Two nucleoside diphosphate analogs, 3'-C-methyl-ADP and 3'-C-methyl-UDP, have been tested as substrate and/or allosteric effectors using the adenosylcobalamin-dependent ribonucleoside diphosphate reductase of Corynebacterium nephridii. Neither analog was a substrate for the reductase. However, they did function as allosteric effectors and as inhibitors of the reduction of ADP and UDP, respectively. The nucleotide analogs did not stimulate the hydrogen exchange reaction between [5'-3H2]adenosylcobalamin and the solvent, indicating that the cleavage of the 3'-carbon-hydrogen bond is a prerequisite for the exchange reaction. A reinvestigation of the requirements for the exchange reaction revealed that the deoxyribonucleoside diphosphate products are very effective promoters of this reaction. Indeed, the deoxyribonucleoside diphosphates were found to be more effective in promoting the exchange reaction than the ribonucleoside diphosphate substrates. In contrast, the deoxyribonucleoside triphosphate effectors, dATP, dUTP, and dTTP, were only marginally effective as promoters of this reaction.