Catalysis by ribonuclease A is limited by the rate of substrate association

The value of k(cat)/K(M) for catalysis of RNA cleavage by ribonuclease (RNase) A can exceed 10(9) M(-1) s(-1) in a solution of low salt concentration. This value approaches that expected for the diffusional encounter of the enzyme and its substrate. To reveal the physicochemical constraints upon catalysis by RNase A, the effects of salt concentration, pH, solvent isotope, and solvent viscosity on catalysis were determined with synthetic substrates that bind to all of the enzymic subsites and thereby enable a meaningful analysis. The pK(a) values determined from pH-k(cat)/K(M) profiles at 0.010, 0.20, and 1.0 M NaCl are inconsistent with the known macroscopic pK(a) values of RNase A. This incongruity indicates that catalysis of RNA cleavage by RNase A is limited by the rate of substrate association, even at 1.0 M NaCl. The effect of solvent isotope and solvent viscosity on catalysis support this conclusion. The data are consistent with a mechanism in which RNase A associates with RNA in an intermediate complex, which is stabilized by Coulombic interactions, prior to the formation of a Michaelis complex. Thus, RNase A has evolved to become an enzyme limited by physics rather than chemistry, a requisite attribute of a perfect catalyst.     

Limits to Catalysis by Ribonuclease A

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of the P-O(5') bond in RNA. Although this enzyme has been the object of much landmark work in bioorganic chemistry, the nature of its rate-limiting transition state and its catalytic rate enhancement had been unknown. Here, the value of k(cat)/K(m) for the cleavage of UpA by wild-type RNase A was found to be inversely related to the concentration of added glycerol. In contrast, the values of k(cat)/K(m) for the cleavage of UpA by a sluggish mutant of RNase A and the cleavage of the poor substrate UpOC(6)H(4)-p-NO(2) by wild-type RNase A were found to be independent of glycerol concentration. Yet, UpA cleavage by the wild-type and mutant enzymes was found to have the same dependence on sucrose concentration, indicating that catalysis of UpA cleavage by RNase A is limited by desolvation. The rate of UpA cleavage by RNase A is maximal at pH 6.0, where k(cat) = 1.4 × 10(3) s(-1) and k(cat)/K(m) = 2.3 × 10(6) M(-1)s(-1) at 25°C. At pH 6.0 and 25°C, the uncatalyzed rate of [5,6-(3)H]Up[3,5,8-(3)H]A cleavage was found to be k(uncat) = 5 × 10(-9) s(-1) (t(1/2) = 4 years). Thus, RNase A enhances the rate of UpA cleavage by 3 × 10(11)-fold by binding to the transition state for P-O(5') bond cleavage with a dissociation constant of <2 × 10(-15) M.     

Potent inhibition of ribonuclease A by oligo(vinylsulfonic acid)

Ribonuclease A (RNase A) can make multiple contacts with an RNA substrate. In particular, the enzymatic active site and adjacent subsites bind sequential phosphoryl groups in the RNA backbone through Coulombic interactions. Here, oligomers of vinylsulfonic acid (OVS) are shown to be potent inhibitors of RNase A that exploit these interactions. Inhibition is competitive with substrate and has Ki = 11 pm in assays at low salt concentration. The effect of salt concentration on inhibition indicates that nearly eight favorable Coulombic interactions occur in the RNase A.OVS complex. The phosphonic acid and sulfuric acid analogs of OVS are also potent inhibitors although slightly less effective. OVS is also shown to be a contaminant of MES and other buffers that contain sulfonylethyl groups. Oligomers greater than nine units in length can be isolated from commercial MES buffer. Inhibition by contaminating OVS is responsible for the apparent decrease in catalytic activity that has been observed in assays of RNase A at low salt concentration. Thus, OVS is both a useful inhibitor of RNase A and a potential bane to chemists and biochemists who use ethanesulfonic acid buffers.     

Decavanadate inhibits catalysis by ribonuclease A

Pentavalent organo-vanadates have been used extensively to mimic the transition state of phosphoryl group transfer reactions. Here, decavanadate (V(10)O(28)6-) is shown to be an inhibitor of catalysis by bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry shows that the Kd for the RNase A decavanadate complex is 1.4 microM. This value is consistent with kinetic measurements of the inhibition of enzymatic catalysis. The interaction between RNase A and decavanadate has a coulombic component, as the affinity for decavanadate is diminished by NaCl and binding is weaker to variant enzymes in which one (K41A RNase A) or three (K7A/R10A/K66A RNase A) of the cationic residues near the active site have been replaced with alanine. Decavanadate is thus the first oxometalate to be identified as an inhibitor of catalysis by a ribonuclease. Surprisingly, decavanadate binds to RNase A with an affinity similar to that of the pentavalent organo-vanadate, uridine 2',3'-cyclic vanadate.     

Ribonuclease III cleavage of a bacteriophage T7 processing signal. Divalent cation specificity, and specific anion effects.

Escherichia coli ribonuclease III, purified to homogeneity from an overexpressing bacterial strain, exhibits a high catalytic efficiency and thermostable processing activity in vitro. The RNase III-catalyzed cleavage of a 47 nucleotide substrate (R1.1 RNA), based on the bacteriophage T7 R1.1 processing signal, follows substrate saturation kinetics, with a Km of 0.26 microM, and kcat of 7.7 min.-1 (37 degrees C, in buffer containing 250 mM potassium glutamate and 10 mM MgCl2). Mn2+ and Co2+ can support the enzymatic cleavage of the R1.1 RNA canonical site, and both metal ions exhibit concentration dependences similar to that of Mg2+. Mn2+ and Co2+ in addition promote enzymatic cleavage of a secondary site in R1.1 RNA, which is proposed to result from the altered hydrolytic activity of the metalloenzyme (RNase III 'star' activity), exhibiting a broadened cleavage specificity. Neither Ca2+ nor Zn2+ support RNase III processing, and Zn2+ moreover inhibits the Mg(2+)-dependent enzymatic reaction without blocking substrate binding. RNase III does not require monovalent salt for processing activity; however, the in vitro reactivity pattern is influenced by the monovalent salt concentration, as well as type of anion. First, R1.1 RNA secondary site cleavage increases as the salt concentration is lowered, perhaps reflecting enhanced enzyme binding to substrate. Second, the substitution of glutamate anion for chloride anion extends the salt concentration range within which efficient processing occurs. Third, fluoride anion inhibits RNase III-catalyzed cleavage, by a mechanism which does not involve inhibition of substrate binding.     

The ribonucleolytic activity of angiogenin.

Angiogenin (ANG), a homologue of bovine pancreatic ribonuclease A (RNase A), promotes the growth of new blood vessels. The biological activity of ANG is dependent on its ribonucleolytic activity, which is far lower than that of RNase A. Here, the efficient heterologous production of human ANG in Escherichia coli was achieved by replacing two sequences of rare codons with codons favored by E. coli. Hypersensitive fluorogenic substrates were used to determine steady-state kinetic parameters for catalysis by ANG in continuous assays. The ANG pH-rate profile is a classic bell-shaped curve, with pK(1) = 5.0 and pK(2) = 7.0. The ribonucleolytic activity of ANG is highly sensitive to Na(+) concentration. A decrease in Na(+) concentration from 0.25 to 0.025 M causes a 170-fold increase in the value of k(cat)/K(M). Likewise, the binding of ANG to a tetranucleotide substrate analogue is dependent on [Na(+)]. ANG cleaves a dinucleotide version of the fluorogenic substrates with a k(cat)/K(M) value of 61 M(-1) s(-1). When the substrate is extended from two nucleotides to four or six nucleotides, values of k(cat)/K(M) increase by 5- and 12-fold, respectively. Together, these data provide a thorough picture of substrate binding and turnover by ANG.     

Correlation between catalysis and tertiary structure arrangement in an archaeal halophilic subtilase

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon N. magadii that exhibits optimal activity and stability in salt-saturated solutions. In this work, the effect of salt on the function and structure of Nep was investigated. In absence of salt, Nep became unfolded and aggregated, leading to the loss of activity. The enzyme did not recover its structural and functional properties even after restoring the ideal conditions for catalysis. At salt concentrations higher than 1 M (NaCl), Nep behaved as monomers in solution and its enzymatic activity displayed a nonlinear concave-up dependence with salt concentration resulting in a 20-fold activation at 4 M NaCl. Although transition from a high to a low-saline environment (3–1 M NaCl) did not affect its secondary structure contents, it diminished the enzyme stability and provoked large structural rearrangements, changing from an elongated shape at 3 M NaCl to a compact conformational state at 1 M NaCl. The thermodynamic analysis of peptide hydrolysis by Nep suggests a significant enzyme reorganization depending on the environmental salinity, which supports in solution SAXS and DLS studies. Moreover, solvent kinetic isotopic effect (SKIE) data indicates the general acid-base mechanism as the rate-limiting step for Nep catalysis, like classical serine-peptidases. All these data correlate the Nep conformational states with the enzymatic behavior providing a further understanding on the stability and structural determinants for the functioning of halolysins under different salinities.     

Pre-steady-state and stopped-flow fluorescence analysis of Escherichia coli ribonuclease III: insights into mechanism and conformational changes associated with binding and catalysis

To better understand substrate recognition and catalysis by RNase III, we examined steady-state and pre-steady-state reaction kinetics, and changes in intrinsic enzyme fluorescence. The multiple turnover cleavage of a model RNA substrate shows a pre-steady-state burst of product formation followed by a slower phase, indicating that the steady-state reaction rate is not limited by substrate cleavage. RNase III catalyzed hydrolysis is slower at low pH, permitting the use of pre-steady-state kinetics to measure the dissociation constant for formation of the enzyme-substrate complex (K(d)=5.4(+/-0.6) nM), and the rate constant for phosphodiester bond cleavage (k(c)=1.160(+/-0.001) min(-1), pH 5.4). Isotope incorporation analysis shows that a single solvent oxygen atom is incorporated into the 5' phosphate of the RNA product, which demonstrates that the cleavage step is irreversible. Analysis of the pH dependence of the single turnover rate constant, k(c), fits best to a model for two or more titratable groups with pK(a) of ca 5.6, suggesting a role for conserved acidic residues in catalysis. Additionally, we find that k(c) is dependent on the pK(a) value of the hydrated divalent metal ion included in the reaction, providing evidence for participation of a metal ion hydroxide in catalysis, potentially in developing the nucleophile for the hydrolysis reaction. In order to assess whether conformational changes also contribute to the enzyme mechanism, we monitored intrinsic tryptophan fluorescence. During a single round of binding and cleavage by the enzyme we detect a biphasic change in fluorescence. The rate of the initial increase in fluorescence was dependent on substrate concentration yielding a second-order rate constant of 1.0(+/-0.1)x10(8) M(-1) s(-1), while the rate constant of the second phase was concentration independent (6.4(+/-0.8) s(-1); pH 7.3). These data, together with the unique dependence of each phase on divalent metal ion identity and pH, support the hypothesis that the two fluorescence transitions, which we attribute to conformational changes, correlate with substrate binding and catalysis.     

Isolation OP DNA Prom Yeast by Chromatography on Hydroxyapatite

A simple procedure is described for isolation of purified non degraded total DNA from yeast cells. The procedure involves conversion of the cells into sphero-plasts by enzymatic treatment, lysis of the sphero-plasts in 8 M urea - 0.24 M sodium phosphate buffer -0.01 M EDTA (ethylendiamintetraacetic acid, sodium salt) - 1% SDS (sodium dodecyl sulphate), deproteiniza-tion of the lysate with chloroform-phenol and separation of the DNA from proteins, RNA and other contaminants by hydroxyapatite chromatography. The yield is about 90% of the DNA in the starting material (sphero-plasts).     

Effects of monomethoxypoly(ethylene glycol) modification of ribonuclease on antibody recognition, substrate accessibility and conformational stability

The effects of modification of bovine pancreatic ribonuclease A by monomethoxypoly(ethylene glycol) (MPEG) were examined for changes in recognition by antiRNase antibodies, enzymatic activity against low and high molecular weight substrates and conformational stability to temperature elevation. Modified forms of RNase were prepared containing an average of 4, 9, and 11 mol of MPEG/mol protein, by amino group modification. These were analysed by binding to RNase antibodies crosslinked to solid phase-immobilized protein A. The affinity column was incorporated into a high performance liquid chromatograph and the RNase species were studied by both zonal and frontal analytical affinity chromatography. An antibody dissociation constant of 7.6 x 10(-8) M was found for unmodified RNase, as compared to values of 1.3 x 10(-7) and 1.2 x 10(-6) M for RNase with 4 and 9 covalently bound MPEG chains, respectively. Modification also led to progressive loss of enzymatic activity against RNA, down to 3% for the most highly modified enzyme. In contrast, enzymatic activity against cytidine-2',3'-cyclic monophosphate was suppressed to a maximum of only 33% at the highest modification level, and the stability to temperature, as followed by circular dichroism, was reduced only partially, from 67 degrees C for native protein to 57 degrees C for RNase with 11 mol equivalents MPEG incorporated. The above differential effects on enzymatic activity, antibody binding and temperature effects are consistent with the view that MPEG modification has relatively small effects on conformational stability and small molecule accessibility, but more dramatic effects on large molecule (substrate as well as antibody) accessibility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the biochemical properties of recombinant ribonuclease III.

An Escherichia coli double strand specific endoribonuclease, RNase III, was cloned, expressed in large amounts, and purified to homogeneity. Enzyme activity was monitored by assaying fractions for the ability to correctly process exogenous RNA containing specific RNase III cleavage sites. DEAE-Sepharose ion exchange chromatography in the presence of a linear KCl gradient (from 0.02 M to 0.75 M) demonstrated that RNase III exists as two distinct forms. One form elutes at a KCl concentration of 0.13 M and the other elutes at 0.33 M. The presence of stoichiometric amounts of the GTP-binding protein Era during purification results in the conversion of the low salt form into the high salt form. Size exclusion chromatography demonstrated that both forms exist as a dimer in solution. In order to investigate the nature of the dimer, protein cross-linking was performed and cross-linked products were detected by silver staining. The protein-protein dimer can be visualized at protein:cross-linker molar ratios as low as 1:15 within 1 minute of exposure to cross-linker in 0.1 M KCl. Upon addition of substrate RNA to the cross-linking reaction a second form of the protein-protein dimer (with a slightly smaller apparent molecular weight) becomes prominent. Induction of the new form is absolutely dependent upon the addition of substrate mRNA to the reaction mixture. We postulate that the RNase III dimer undergoes a dramatic conformational change upon recognition of RNA which we are able to trap by cross-linking.     

RNase A effects on sedimentation and DNA binding properties of dexamethasone-receptor complexes from HeLa cell cytosol

Dexamethasone-receptor complexes from HeLa cell cytosol sediment at 7.4S in low salt sucrose gradients, and at 3.8S in high salt gradients. If cytosol is heated at 25 degrees C, receptor complexes sediment at 6.9S in low salt, and at 3.6S in high salt gradients. RNase A treatment at 25 degrees C, instead, results in receptor complexes which sediment in low salt gradients as two major forms at 6.5 and 4.8S. Receptor complexes from RNase A-treated cytosols sediment as their counterparts from untreated cytosols in high salt gradients. Although the shift in sedimentation properties of receptor complexes at 2 degrees C is induced by RNase A, and not by other low molecular weight basic proteins or RNase T1, the effect can be also obtained by inactive RNase A. The catalytically active enzyme, however, is required to observe 6.5 and 4.8S complexes after cytosol incubations at 25 degrees C. Placental ribonuclease inhibitor prevents the appearance of RNase A-induced receptor forms at 25 degrees C, but not at 2 degrees C. Moreover, this inhibitor can prevent the 7.4 to 6.9S shift in sedimentation coefficient of receptor complexes caused by cytosol heating. Dexamethasone-receptor complexes from HeLa cell cytosol show low levels of binding to DNA-cellulose, and heating at 25 degrees C is required to observe a six-fold increase in DNA binding levels. RNase A treatment of cytosols at 2 degrees C does not result in significant enhancement in receptor complex binding to DNA. If RNase A treatment is carried out at 25 degrees C, however, DNA binding levels of receptor complexes increased by 25% over the values observed with control heated cytosol. This effect cannot be observed if RNase T1 substitutes for RNase A. Placental ribonuclease inhibitor can prevent the temperature-dependent increase in DNA binding properties of dexamethasone-receptor complexes either in the presence or absence of exogenous RNase A. These findings indicate that exogenous RNases can perturb the structure of dexamethasone-receptor complexes without being involved in the transformation process.     

Allosteric control of cAMP receptor binding dynamics

The intrinsic fluorescence of the cyclic AMP receptor is a sensitive indicator of the reaction with DNA, but signals are perturbed by a photoreaction. A ratio procedure is shown to be useful for correction. The reaction of the protein with DNA indicated by corrected transients extends over a broad time range not only at low salt concentrations but also at physiological salt concentrations. The initial binding step can be recorded preferentially at low salt pH 7 and is shown to be very similar for specific and nonspecific DNA. The rate constant for initial binding at 13.5 mM salt pH 7 is 2 × 10(8) M(-1) s(-1). Slow reaction steps up to times of several hundred seconds are observed both at low and high salt; the magnitude and sign of fluorescence amplitudes are strongly dependent on salt and pH. At 100 mM salt pH 8, the slow reaction step observed for the binding of the cyclic AMP receptor protein to promoter DNA is strongly shifted to longer times upon reduction of the cAMP concentration. The observed cAMP dependence is described quantitatively by a model implying that binding of the receptor to promoter DNA requires two cAMP molecules per protein dimer and is not consistent with a model assuming that a single cAMP is sufficient for activation. The rate constant for binding of the protein·dimer·(cAMP)(2) complex to the promoter is 1.3 × 10(8) M(-1) s(-1), close to the limit of diffusion control. Equilibration of specific complexes takes ~100 s at physiological concentrations of the reaction components.     

Interaction of cisplatin drug with RNase A.

cis-Pt(NH3)2Cl2 (cisplatin) is an antitumor drug with many severe toxic side effects including enzymatic structural changes associated with its mechanism of action. This study is designed to examine the interaction of cisplatin drug with ribonuclease A (RNase A) in aqueous solution at physiological pH, using drug concentration of 0.0001 mM to 0.1 mM with final protein concentration of 2% w/v. Absorption spectra and Fourier transform infrared (FTIR) spectroscopy with its self-deconvolution, second derivative resolution enhancement and curve-fitting procedures were used to characterize the drug binding mode, association constant and the protein secondary structure in the cisplatin-RNase complexes. Spectroscopic results show that at low drug concentration (0.0001 mM), no interaction occurs between cisplatin and RNase, while at higher drug concentrations, cisplatin binds indirectly to the polypeptide C=O, C-N (via H2O or NH3 group) and directly to the S-H donor atom with overall binding constant 5.66 x 10(3)M(-1). At high drug concentration, major protein secondary structural changes occur from that of the alpha-helix 29% (free enzyme) to 20% and beta-sheet 39% (free enzyme) to 45% in the cisplatin-RNase complexes. The observed structural changes indicate a partial protein unfolding in the presence of cisplatin at high drug concentration.     

On the interaction of bovine seminal RNase with actin in vitro

Ribonuclease from bovine seminal plasma (RNase BS) interacts with skeletal muscle actin in the following way: it binds to actin with an apparent binding constant of 9.2 X 10(4) M-1 in 0.1 M KCl, induces the polymerization of actin below the critical concentration in depolymerization buffer, accelerates the salt-induced polymerization of actin even at a molar ratio of RNase to actin lower than 1/100, and bundles F-actin filaments. In the bundles the molar ratio of RNase to actin is about 0.66. Actin inhibits the enzymatic activity of RNase BS. RNase A from bovine pancreas, which is structurally almost identical to the subunits of RNase BS as well as a monomeric form of RNase BS, do not cross-link actin filaments and have a much smaller effect on the polymerization of actin. We conclude that the dimeric structure of the RNase BS, which consists of two identical subunits cross-linked by interchain disulfide bridges, is probably responsible for the bundling activity and the accelerating effect on the polymerization of actin.     

Expression of human placental ribonuclease inhibitor in Escherichia coli

Human placental ribonuclease inhibitor (PRI) has been expressed in and isolated from Escherichia coli. Its apparent molecular weight, immunoreactivity and amino acid composition are virtually identical with those of native PRI. It inhibits the enzymatic activities of either angiogenin, a blood vessel inducing protein homologous to pancreatic RNase (RNase A), or RNase A in a stoichiometry of 1:1. Recombinant PRI binds to angiogenin and RNase A with Ki values of 2.9 x 10(-16) M and 6.8 x 10(-14) M, respectively, comparable to the affinities of native PRI for these enzymes. Thus, these results confirm that PRI inhibits angiogenin more effectively than RNase A.     

Broad-specificity endoribonucleases and mRNA degradation in Escherichia coli.

Crude extracts from Escherichia coli were screened for any broad-specificity endoribonuclease after the cell proteins were fractionated by size. In a mutant lacking the gene for RNase I (molecular mass, 27,156 Da), the only such activities were also in the size range of 23 to 28 kDa. Fractionation by chromatography on a strong cation-exchange resin revealed only two activities. One of them eluted at a salt concentration expected for RNase M and had the specificity of RNase M. It preferred pyrimidine-adenosine bonds, could not degrade purine homopolymers, and had a molecular mass of approximately 27 kDa (V. J. Cannistraro and D. Kennell, Eur. J. Biochem. 181:363-370, 1989). A second fraction, eluting at a higher salt concentration, was active against any phosphodiester bond but was about 100 times less active than are RNase I and RNase I* (a form of RNase I) in the wild-type cell. On the basis of sizing-gel chromatography, this enzyme had a molecular mass of approximately 24 kDa. We call it RNase R (for residual). RNase R is not an abnormal product of the mutant rna gene; a cell carrying many copies of that gene on a plasmid did not synthesize more RNase R. Our search for broad-specificity endoribonucleases was prompted by the expectation that the primary activities for mRNA degradation are expressed by a relatively small number of broad-specificity RNases. If correct, the results suggest that the endoribonucleases for this major metabolic activity reside in the 24- to 28-kDa size range. Endoribonucleases with much greater specificity must have as primary functions the processing of specific RNA molecules at a very limited number of sites as steps in their biosynthesis. In exceptional cases, these endoribonucleases inactivate a specific message that has such a site, and they can also effect total mRNA metabolism indirectly by a global disturbance of the cell physiology. It is suggested that a distinction be made between these processing and degradative activities.     

Effects of phosphorothioate modifications on precursor tRNA processing by eukaryotic RNase P enzymes

The cleavage mechanism has been studied for nuclear RNase P from Saccharomyces cerevisiae, Homo sapiens sapiens and Dictyostelium discoideum, representing distantly related branches of the Eukarya. This was accomplished by using precursor tRNAs (ptRNAs) carrying a single Rp or Sp-phosphorothioate modification at the normal RNase P cleavage site (position -1/+1). All three eukaryotic RNase P enzymes cleaved the Sp-diastereomeric ptRNA exclusively one nucleotide upstream (position -2/-1) of the modified canonical cleavage site. Rp-diastereomeric ptRNA was cleaved with low efficiency at the modified -1/+1 site by human RNase P, at both the -2/-1 and -1/+1 site by yeast RNase P, and exclusively at the -2/-1 site by D. discoideum RNase P. The presence of Mn(2+ )and particularly Cd(2+) inhibited the activity of all three enzymes. Nevertheless, a Mn(2+ )rescue of cleavage at the modified -1/+1 site was observed with yeast RNase P and the Rp-diastereomeric ptRNA, consistent with direct metal ion coordination to the (pro)-Rp substituent during catalysis as observed for bacterial RNase P enzymes. In summary, our results have revealed common active-site constraints for eukaryotic and bacterial RNase P enzymes. In all cases, an Rp as well as an Sp-phosphorothioate modification at the RNase P cleavage site strongly interfered with the catalytic process, whereas substantial functional interference is essentially restricted to one of the two diastereomers in other RNA and protein-catalyzed hydrolysis reactions, such as those catalyzed by the Tetrahymena ribozyme and nuclease P1.     

Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatically synthesized 2',5'-phosphorothioate dimer and trimer: unequivocal structural assignment and activation of 2',5'-oligoadenylate-dependent endoribonuclease

In continued studies to elucidate the requirements for binding to and activation of the 2',5'-oligoadenylate-dependent endoribonuclease (RNase L), chirality has been introduced into the 2',5'-oligoadenylate (2-5A, p3An) molecule to give the Rp configuration in the 2',5'-internucleotide backbone and the Sp configuration in the alpha-phosphorus of the pyrophosphoryl moiety of the 5'-terminus. This was accomplished by the enzymatic conversion of (Sp)-ATP alpha S to the 2',5'-phosphorothioate dimer and trimer by the 2-5A synthetase from lysed rabbit reticulocytes. The most striking finding reported here is the ability of the 2',5'-phosphorothioate dimer 5'-triphosphate (i.e., p3A2 alpha S) to bind to and activate RNase L. p3A2 alpha S displaces the p3A4[32P]pCp probe from RNase L with an IC50 of 5 X 10(-7) M, compared to an IC50 of 5 X 10(-9) M for authentic p3A3. Further, p3A2 alpha S activates RNase L to hydrolyze poly(U)-3'-[32P]pCp (20% at 2 X 10(-7) M), whereas authentic p3A2 is unable to activate the enzyme. Similarly, the enzymatically synthesized p3A2 alpha S at 10(-6) M activated RNase L to degrade 18S and 28S rRNA, whereas authentic p3A2 was devoid of activity. p3A3 alpha S was as active as authentic p3A3 in the core--cellulose and rRNA cleavage assays. The absolute structural and configurational assignment of the enzymatically synthesized p3A2 alpha S and p3A3 alpha S was accomplished by high-performance liquid chromatography, charge separation, enzymatic hydrolyses, and comparison to fully characterized chemically synthesized (Rp)- and (Sp)-2', 5'-phosphorothioate dimer and trimer cores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis.

The rnhA gene encoding RNase HI from a psychrotrophic bacterium, Shewanella sp. SIB1, was cloned, sequenced and overexpressed in an rnh mutant strain of Escherichia coli. SIB1 RNase HI is composed of 157 amino acid residues and shows 63% amino acid sequence identity to E.coli RNase HI. Upon induction, the recombinant protein accumulated in the cells in an insoluble form. This protein was solubilized and purified in the presence of 7 M urea and refolded by removing urea. Determination of the enzymatic activity using M13 DNA-RNA hybrid as a substrate revealed that the enzymatic properties of SIB1 RNase HI, such as divalent cation requirement, pH optimum and cleavage mode of a substrate, are similar to those of E.coli RNase HI. However, SIB1 RNase HI was much less stable than E.coli RNase HI and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was approximately 25 degrees C for SIB1 RNase HI and approximately 60 degrees C for E.coli RNase HI. The optimum temperature for the SIB1 RNase HI activity was also shifted downward by 20 degrees C compared with that of E.coli RNase HI. Nevertheless, SIB1 RNase HI was less active than E.coli RNase HI even at low temperatures. The specific activity determined at 10 degrees C was 0.29 units/mg for SIB1 RNase HI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesis studies suggest that the amino acid substitution in the middle of the alphaI-helix (Pro52 for SIB1 RNase HI and Ala52 for E.coli RNase HI) partly accounts for the difference in the stability and activity between SIB1 and E.coli RNases HI.