Study of ATP-independent stages of reaction catalyzed by phage T4 RNA-ligase

The isotope exchange between [5'-32P]pAP and A(5')ppAp catalyzed by enzyme was shown not to take place in the absence of the acceptor; i. e. the necessity of the acceptor presence during the second step of the process was demonstrated. The isotope exchange reaction between [5'32P]pAp and (pA)5p was studied. It was demonstrated that acceptor (pA)4, slightly whereas the acceptor (pU)4 completely inhibits the isotope reaction. The isotope reaction exchange between [5'-32P]pAp and (pU)4pAp does not take place. The question of existence of adenylated donor elimination mechanism in the presence of "poor" acceptors is considered on the basis of the data obtained.     

RNA-ligase of bacteriophage T4. IV. Synthesis of pentadecaribonucleotide ApUpG(pU)6(pA)5pAp

Oligoribonucleotide ApUpG(pU)6(pA)5pAp (I) has been prepared by means of RNA ligase. ApUpG, synthesised by the phosphotriester approach, was elongated in the 3'-direction by adding (pU)6 and then (pA)6, which was 3'-blocked with the phosphate or with the periodate-oxidized AMP residue, the latter giving considerably lower level of by-products. Condensation of ApUpG(pU)6 with blocked (pA)6 was carried out under conditions optimal for poor acceptors (20 degrees C, 48 h, pH 8,7) to afford (I) with the yield of 20% (105 OE260); ApUpG(pU)6(pA)10pAp was identified as a byproduct.     

The mode of Mg++ binding to oligonucleotides. Inner sphere complexes as markers for recognition?

Large changes of UV absorbance and CD spectra as well as specific relaxation processes with time constrants around 50 mus are found for the association of Mg++ with A(pa)n. The Mg++ binding constants strongly increase with increasing n. The relaxation data demonstrate that a large fraction of Mg++ bound to short A(pA)n forms inner sphere complexes (ISC), with H2O molecules from the inner hydration sphere of Mg++ exchanged against some site (s) of the oligomer. This fraction decreases from about 85% for A(pA)4 to less than 10% for A(pA)17. A parallel decrease is observed in the relative change of CD spectrum upon Mg++ binding from 77.5% for A(pA)4 to 13.4% for (pA)17. The rate of ISC formation decreases with increasing n suggesting some (probably sterical) hindrance effect at high n. The data support the conclusion that Mg++ favours the formation of outer sphere complexes with linear polynucleotides and require a special chain folding for ISC. Measurements of Mg++ binding to C(pC)5, U(pU)5, I(pI)5 and d[A(pA)5] did not give evidence for the formation of ISC, indicating that both specific base and sugar residues are required for ISC. These results suggest the possibility that Mg++ISC ARE USED FOR SPECific recognition of nucleic acid sequences.     

Studies of oligoadenylate formation on a poly(U) template

We have studied a variety of condensation reactions involving poly (U) as template and isomeric adenosine dinucleotides as substrates. We find that [3'-5']-linked dinucleotides such as A3pA and pA3pA are better acceptors than the corresponding [2'-5']-linked compounds, while ImpA2pA is a better donor than ImpA3pA. The reaction between A2pA and ImpA3pA, for example, yields only 4% of product while the reaction of A3pA with ImpA2pA yields 86% of product. The more efficient condensation reactions of dimers are about as efficient as the self-condensation of ImpA. They yield a few percent of material in which five or more substrate molecules are linked together. The percentage of the natural [3'-5']-linkage in the product varies greatly, from as little as 1% to as much as 45%.     

Bacteriophage T4 RNA ligase. Reaction intermediates and interaction of substrates.

Bacteriophage T4 RNA ligase catalyzes the ATP-dependent ligation of a 5'-phosphoryl-terminated nucleic acid donor to a 3'-hydroxyl-terminated nucleic acid acceptor. We have identified adenylylated DNA and RNA reaction intermediates in which the AMP moiety is attached by a pyrophosphate bond to the 5'-phosphoryl group of the donor. A large amount of DNA-adenylate accumulates during the reaction and the dependence of joining and adenylylation on chain length are similar. The adenylylated donor is joined by ligase to an acceptor in the absence of ATP, and AMP is released stoichiometrically in this reaction. The acceptor is not only a substrate in the reaction but also a cofactor for adenylylation of the donor; in the absence of a 3'-hydroxyl group the activated intermediate does not form. The activated DNA need not join to the acceptor that initially stimulated activation but can also join to another acceptor. This process of acceptor exchanges has proven useful for promoting the cyclization of small DNA substrates and the synthesis of DNA co-polymers.     

Several dinucleoside polyphosphates are acceptor substrates in the T4 RNA ligase catalyzed reaction.

Several dinucleoside polyphosphates accept cytidine-3', 5'-bisphosphate from the adenylylated donor 5'-adenylylated cytidine 5',3'-bisphosphate in the T4 RNA ligase catalyzed reaction. The 5'-adenylylated cytidine 5',3'-bisphosphate synthesized in a first step, from ATP and cytidine-3',5'-bisphosphate, is used as a substrate to transfer the cytidine-3',5'-bisphosphate residue to the 3'-OH group(s) of diguanosine tetraphosphate (Gp4G) giving rise to Gp4GpCp and pCpGp4GpCp in a ratio of approximately 10 : 1, respectively. The synthesized Gp4GpCp was characterized by treatment with snake venom phosphodiesterase and alkaline phosphatase and analysis (chromatographic position and UV spectra) of the reaction products by HPLC. The apparent Km values measured for Gp4G and 5'-adenylylated cytidine 5',3'-bisphosphate in this reaction were approximately 4 mM and 0.4 mM, respectively. In the presence of 0.5 mM ATP and 0.5 mM cytidine-3',5'-bisphosphate, the relative efficiencies of the following nucleoside(5')oligophospho(5')nucleosides as acceptors of cytidine-3',5'-bisphosphate from 5'-adenylylated cytidine 5', 3'-bisphosphate are indicated in parentheses: Gp4G (100); Gp5G (101); Ap4G (47); Ap4A (39). Gp2G, Gp3G and Xp4X were not substrates of the reaction. Dinucleotides containing two guanines and at least four inner phosphates were the preferred acceptors of cytidine-3', 5'-bisphosphate at their 3'-OH group(s).     

Helical Structure Formation Between Complementary Oligonucleotides. Minimum Chain Length Required for the Template-Directed Synthesis of Oligonucleotides

Helix formation between various combinations of 3–5 linked oligoribouridylates and oligoriboadenylates from dimer to dodecamer has been studied to gain information on the chain-length requirement for the template-directed condensation of oligoribonucleotides. We have measured the helix formation under high oligoribonucleotide concentration in the presence of magnesium ion at 0–50°C by UV or CD, as many model processes of oligoribonucleotides replication have been carried out under such conditions. Adenylic acid, (pA), diadenylic acid, (pA)₂, or triadenylic acid, (pA)₃, forms a helix with poly(U) or oligo(U) with a chain length of more than eight. On the other hand, neither uridylic acid, (pU), nor diuridylic acid, (pU)₂, can form a helix with oligo(A) or poly(A). Triuridylic acid, (pU)₃, or the longer oligo(U) forms a helix with oligo(A) with a chain length of over six. The results suggest that a trimer is the minimum unit as an incorporating nucleotide for conducting any set of nonenzymatic template-directed synthesis, AU and UA, as the nonenzymatic template-directed condensation of oligoribonucleotides correlates well with the results of helix formation of complementary oligoribonucleotides. We have further found the partial helix formation between 2–5 linked decauridylate, (pU)₁₀, and pA or 2–5 linked (pA)₂ at 0 °C, which indicates the possibility of the template activity of long 2–5 linked oligonucleotides for the nonenzymatic oligonucleotide synthesis.     

The halide complexes of myeloperoxidase and the mechanism of the halogenation reactions

The spectral changes caused by the addition of halides to myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) have been investigated and the dissociation constants of the enzyme-halide complexes have been determined. The pH dependence of the dissociation constants suggests that halide binding is associated with a protonation step in myeloperoxidase. Myeloperoxidase catalyzes the peroxidative chlorination and bromination of monochlorodimedone. It is shown that at low pH, chloride acts as a competitive inhibitor with respect to H2O2, whereas at higher pH, H2O2 inhibits the chlorination reaction. The dissociation constant (Kd) of the spectroscopically detectable complex and the Km for chloride are considerably smaller than the inhibition constant (Ki) for chloride. These halogenation reactions are strongly pH dependent, the logarithm of the Km for chloride varies linearly with pH. The position of the pH optimum of the chlorination and bromination reaction is a linear function of the logarithm of the [halide]/[H2O2] ratio. A mechanism of the chlorination and bromination reaction is suggested with substrate inhibition for both hydrogen peroxide and the halide.     

Phosphoserine and specific types of its coordination in copper(II) and adenosine nucleotides systems - Potentiometric and spectroscopic studies

Ternary systems of copper(II) complexes with phosphoserine and adenosine 5′-monophosphate or adenosine 5′-diphosphate or adenosine 5′-triphosphate have been investigated. The studies have been performed in aqueous solution using the potentiometric method with computer analysis of the data, ¹³C and ³¹P nuclear magnetic resonance, visible and electron paramagnetic resonance spectroscopies. The overall stability constants of the complexes have been determined. Analysis of the equilibrium constants of the reaction have allowed determination of the effectiveness of the phosphate groups and donor atoms of heterocyclic rings in the process of complex formation. The potential reaction centres are the nitrogen atoms N(1) and N(7) and the phosphate group of the nucleotides as well as phosphate, carboxyl and amine groups from phosphorylated serine. Coordination sites of investigated ligands have been identified on the basis of the equilibrium constants analysis and spectroscopic studies.     

Catalysis and Selectivity in Prebiotic Synthesis: Initiation of the Formation of Oligo(U)s on Montmorillonite Clay by Adenosine-5′-methylphosphate

Adenosine-5′-methylphosphate (MepA) initiates the oligomerization of the 5′-phosphorimidazolide of uridine (ImpU) in the presence of montmorillonite clay. Longer oligomers form because the 5′-phosphate is blocked with a methyl group that prevents the formation of cyclic- and pyrophosphate-containing compounds. The MepA initiates 69–84% of the 5–9 charge oligomers, respectively. The montmorillonite catalyst also provides selectivity in the oligomerization reactions so that the main reaction pathway is MepA → MepA³′pU → MepA³′pU²′pU → MepA³′pU²′pU³′pU. MepA did not enhance the oligomerization of ImpA. The relative rates of the reactions were determined from an investigation of the products in competitive reactions. Selectivity was observed in the reaction of ImpU with equimolar amounts of MepA³′pU and MepA²′pU where the relative reaction rates are 10.3:1, respectively. In the reaction of ImpA with MepA³′pA and MepA²′pA the ImpA reacts 5.2 times faster with MepA³′pA. In the competitive reaction of ImpU and ImpA with MepA³′pA and MepA³′pU the elongation proceeds on MepA³′pA 5.6 times more rapidly than with MepA³′pU. There is no correlation between the extent of binding to the montmorillonite and reaction rates in the formation of longer oligomers. The formation of more than two sequential 2′,5′-linkages in the oligomer chain proceeds more slowly than the addition to a single 2′,5′-link or a 3′,5′-link and either chain termination or elongation by a 3′,5′-linage occurs. The central role that catalysis may have had in the prebiotic formation of biopolymers is discussed. Note added in proof: There are errors in the high resolution mass spectral data given in Section 4.2.1. The high resolution mass spectrum found for the cyclic dimer of UpUp (C-UpUp) was 657.02260. C₁₈H₂₁N₄O₁₆P₂Na₂ requires 657.02232. The high resolution mass spectrum found for the cyclic dimer of ApAp (C-ApAp) was 725.05850. C₂₀H₂₂N₁₀O₁₂P₂Na₃ requires 725.05839.     

The kinetics of complex formation of tryptophan containing proteins with pyridinium salts

Upon preincubation with urea, various 3- or 4-substituted N-methylpyridinium salts form charge-transfer complexes with tryptophan containing proteins such as, L-chymotrypsin and lysozyme. The complexes were studied by using the difference spectrophotometric technique. The fluorescence examination showed that tryptophyl residues in protein molecules are engaged in the complex formation process. The complex formation reactions proceed at a considerable rate. The stopped-flow method was used to determine the pseudo first order rate constants. A linear dependence of the pseudo first order rate constants with the donor concentration was found. The second order rate constants were obtained by dividing the mean value of the pseudo first order rate constants by the initial donor concentration for each run. The linear dependence of second order rate constants with the electron affinity of the acceptors can serve as a criterion for the formation of charge-transfer complexes.     

Kinetics and mechanism in the reaction of gene regulatory proteins with DNA

We have measured the kinetic properties of the Escherichia coli cAMP receptor protein (CAP) and lac repressor interacting with lac promoter restriction fragments. Under our reaction conditions (10 mM-Tris X HCl (pH 8.0 at 21 degrees C), 1 mM-EDTA, 10 microM-cAMP, 50 micrograms bovine serum albumin/ml, 5% glycerol), the association of CAP is at least a two-step process, with an initial, unstable complex formed with rate constant kappa a = 5(+/- 2.5) X 10(7) M-1 s-1. Subsequent formation of a stable complex occurs with an apparent bimolecular rate constant kappa a = 6.7 X 10(6) M-1 s-1. At low total DNA concentration, the dissociation rate constant for the specific CAP-DNA complex is 1.2 X 10(-4) s-1. The ratio of formation and dissociation rate constants yields an estimate of the equilibrium constant, Keq = 5 X 10(10) M-1, in good agreement with static results. We observed that the dissociation rate constant of both CAP-DNA and repressor-DNA complexes is increased by adding non-specific "catalytic" DNA to the reaction mixture. CAP dissociation by the concentration-dependent pathway is second-order in added non-specific DNA, consistent with either the simultaneous or the sequential participation of two DNA molecules in the reaction mechanism. The results imply a role for distal DNA in assembly-disassembly of specific CAP-DNA complexes, and are consistent with a model in which the subunits in the CAP dimer separate in the assembly-disassembly process. The dissociation of lac repressor-operator complexes was found to be DNA concentration-dependent as well, although in contrast to CAP, the reaction is first-order in catalytic DNA. Added excess operator-rich DNA gave more rapid dissociation than equivalent concentrations of non-specific DNA, indicating that the sequence content of the competing DNA influences the rate of repressor dissociation. The simplest interpretation of these observations is that lac repressor can be transferred directly from one DNA molecule to another. A comparison of the translocation rates calculated for direct transfer with those predicted by the one-dimensional sliding model indicates that direct transfer may play a role in the binding site search of lac repressor.     

Reactions of sulfur-nitrosyl iron complexes of "g=2.03" family with hemoglobin (Hb): kinetics of Hb-NO formation in aqueous solutions.

NO-donating ability of nitrosyl [Fe-S] complexes, namely, mononuclear dinitrosyl complexes of anionic type [Fe(S2O3)2(NO)2]-(I) and neutral [Fe2(SL1)2(NO)2] with L1=1H-1,2,4-triazole-3-yl (II); tetranitrosyl binuclear neutral complexes [Fe2(SL2)2(NO)4] with L2=5-amino-1,2,4-triazole-3-yl (III); 1-methyl-1H-tetrazole-5-yl (IV); imidazole-2-yl (V) and 1-methyl-imidazole-2-yl (VI) has been studied. In addition, Roussin's "red salt" Na2[Fe2S2(NO)4] x 8H2O (VII) and Na2[Fe(CN)5NO] x H2O (VIII) have been investigated. The method for research has been based on the formation of Hb-NO adduct upon the interaction of hemoglobin with NO generated by complexes I-VIII in aqueous solutions. Kinetics of NO formation was studied by registration of absorption spectra of the reaction systems containing Hb and the complex under study. For determination of HbNO concentration, the experimental absorption spectra were processed during the reaction using standard program MATHCAD to determine the contribution of individual Hb and HbNO spectra in each spectrum. The reaction rate constants were obtained by analyzing kinetic dependence of Hb interaction with NO donors under study. All kinetic dependences for complexes I-VI were shown to be described well in the frame of formalism of pseudo first-order reactions. The effective first-order rate constants for the studied reactions have been determined. As follows from the values of rate constants, the rate of interaction of sulfur-nitrosyl iron complexes (I-VI) with Hb is limited by the stage of NO release in the solution.     

Kinetic analysis of the mechanism of action of beta-glucosidase from Botryodiplodia theobromae Pat.

1. The kinetic mechanism of beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) of Botryodiplodia theobromae Pat. has been studied in the presence of competing glucosyl acceptors. 2. Glycerol, fructose, sucrose, cellobiose and to a much lesser extent, maltose can act as glucosyl acceptors, apart from water. 3. Evidence confirming and supporting the kinetic mechanism previously postulated (Umezurike, G.M. (1971) Biochim. Biophys. Acta. 250, 182-191) is presented. 4. A theoretical kinetic analysis of the behaviour of the enzyme in the presence of two alternative glucosyl acceptors in addition to water is found to be consistent with experimental observation, suggesting a system in which both donor and acceptors bind to the enzyme in a random fashion to form ternary complexes. 5. The results are discussed in terms of the mechanism of group-transfer reactions.     

Electrometrical study of electron transfer from the terminal FA/FB iron-sulfur clusters to external acceptors in photosystem I

An electrometrical technique was used to investigate electron transfer between the terminal iron-sulfur centers F(A)/F(B) and external electron acceptors in photosystem I (PS I) complexes from the cyanobacterium Synechococcus sp. PCC 6301 and from spinach. The increase of the relative contribution of the slow components of the membrane potential decay kinetics in the presence of both native (ferredoxin, flavodoxin) and artificial (methyl viologen) electron acceptors indicate the effective interaction between the terminal 14Fe-4S] cluster and acceptors. The finding that FA fails to donate electrons to flavodoxin in F(B)-less (HgCl2-treated) PS I complexes suggests that F(B) is the direct electron donor to flavodoxin. The lack of additional electrogenicity under conditions of effective electron transfer from the F(B) redox center to soluble acceptors indicates that this reaction is electrically silent.     

Joining of ribooligonucleotides with T4 RNA ligase and identification of the oligonucleotide-adenylate intermediate.

T4 RNA ligase was found to join A-A-A-A-A-A and 32pU-U-U-U in the presence of ATP as cofactor. In this reaction the pyrophosphate of pU-U-U-U and pA was isolated by chromatography on a RPC-5 column, besides the joined product and the starting materials. This pyrophosphate was shown to be an intermediate in the joining reaction because of the fact that coupling with A-A-A-A-A-A to give the decanucleotide could be performed in the absence of ATP. The structure of the oligonucleotide-adenylate was determined by enzymatic digestion with base-nonspecific nuclease and venom phosphodiesterase. Futher evidence for the proposed structure was obtained by isolation of the intermediate obtained by using pU-U-U-U and [alpha-32p]ATP. This pyrophosphate gave pA and pU by treatment with venom phosphodiesterase. Several other joining reactions between various purine- and pyrimidine ribooligonucleotides to 5'-phosphorylated ribooligonucleotides are discussed.     

Dissociation of guanosine nucleotide-elongation factor G-ribosome complexes.

The spontaneous dissociation of complexes containing elongation factor G (EF-G), the ribosome, and either GDP plus fusidic acid, guanyl-5'-yl imidodiphosphate, or guanyl-5'-yl methylene diphosphonate has been measured and it follows biphasic kinetics that can be resolved into two first-order decay rates. This suggest the existence of two classes of complexes with apparent dissociation rate constants (k) differing 5--20-fold. The values of k and the distribution of complexes between the fast and the slowly decaying class depend on the conditions in which the dissociation occurs but not on the conditions in which the complexes are formed. Rapid transitions of complexes from one to the other class occur only when the chemical environment in which the dissociation takes place is modified. Thus, increasing the concentration of NH4Cl or adding the antibiotic thiostrepton accelerates the decay and converts slowly dissociating into fast dissociating complexes. In contrast, addition of misreading-inducing aminoglycoside antibiotics of the neomycin, kanamycin, streptomycin, and gentamicin (but not hygromycin) groups slows down the decay. For neomycin B at 10 micron, this effect is due to the conversion of fast into slowly decaying complexes. A model to explain the results involving conformational transitions of the complexes is proposed.     

The effect of thiamine pyrophosphate modification on its coenzyme function in a transketolase-catalyzed reaction

The coenzyme function of TPP analogues: 4'-NH-methyl-TPP,6'-methyl-TPP and 6'-methyl-4-nor-TPP has been studied in a transketolase-catalyzed reaction. Their dissociation constants have been found with the aid of the circular dichroism method, and coenzyme activity has been determined in a complete transketolase reaction, involving the substrate-donor and the substrate-acceptor, and also at the intermediate stage (by the alpha-carbanionic intermediate oxidation rate). The coenzyme activity values have been found different and largely dependend on the nature of the substrates used. A possibility of TPP functioning by the "two-center mechanism" in a transketolase-catalyzed reaction is discussed.     

Affinity modification of 80S ribosomes from human placenta by derivatives of tri- and hexauridylates as mRNA analogs]

Derivatives of 5'-32P]labeled (pU)3 and (pU)6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residues attached to 5'-phosphates via phosphamide bond were applied to the affinity labeling of 80S ribosomes from human placenta. The reagents had normal coding properties and were fixed in the ribosomal mRNA-binding region by codon-anticodon interaction with cognate Phe-tRNA(Rhe) at P site (in the case of (pU)3 derivative) or at both A and P sites (in the case of (pU)6 one). Both reagents were found to modify only the 40S subunit. The sites of the reagents attachment to 18S ribosomal RNA were identified by blot-hybridization of the modified 18S rRNA with restriction fragments of the corresponding rDNA. They were found to be located within positions 976-1057 for (pU)6 derivative and within 976-1164 for (pU)3 one. These sites are located presumably within highly conserved parts of the eukaryotic small subunit rRNA secondary structure.