HIV-1 reverse transcriptase interaction with model RNA-DNA duplexes.

HIV-1 reverse transcriptase (HIV-1 RT) is a multifunctional enzyme responsible for converting viral RNA into preintegrative DNA during the early stages of viral infection. DNA polymerase and RNase H activities are required, and several conformationally distinct primer-templates must be accommodated by the enzyme during the process. Parameters of interaction between model substrates (ligands) and HIV-1 RT (wild type p66/p51 and the RNase H-deficient mutant p66(E478Q)/p51) (analytes) were estimated by surface plasmon resonance at 25 degrees C, pH 8.0. Binding of RT to the ligands is specific and can be analyzed using a conventional 1:1 binding algorithm. RNA-DNA hybrids with 5'-template overhangs of 6 and 12 nucleotides bind to RT approximately one order of magnitude stronger than the corresponding 36-mer with blunt ends due to slower dissociation. Immobilization of the latter through either the 5'-end of RNA or DNA strand does not change the equilibrium constant (K(D)) for wild-type RT but the values of kinetic constants of association and dissociation differ significantly. For the p66(E478Q)/p51 enzyme, orientation effects are notable even altering the K(D) value. Binding of the p66(E478Q)/p51 to any RNA-DNA hybrids is slightly stronger compared with wild type. Data can be interpreted in terms of the mechanism of reverse transcription.

Effect of cAMP binding site mutations on the interaction of cAMP receptor protein with cyclic nucleoside monophosphate ligands and DNA.

Although cAMP binding to wild type cAMP receptor protein (CRP) induces specific DNA binding and activates transcription, cyclic nucleoside monophosphate (cNMP) binding to the CRP mutant Ser128 --> Ala does not, whereas the double CRP mutant Thr127 --> Leu/Ser128 --> Ala activates transcription even in the absence of cNMP. Isothermal titration calorimetry measurements on the cNMP binding reactions to the S128A and T127L/S128A mutants show that the reactions are mainly entropically driven as is cAMP binding to CRP. In contrast to cAMP binding to CRP, the binding reactions are noncooperative and exothermic with binding enthalpies (DeltaHb) ranging from -23.4 +/- 0.9 kJ mol-1 for cAMP binding to S128A at 39 degrees C to -4.1 +/- 0.6 kJ mol-1 for cAMP binding to T127L/S128A at 24 degrees C and exhibit enthalpy-entropy compensation. To account for the inactivity of the S128A mutant, in vitro and in vivo DNA binding experiments were performed on the cAMP-ligated S128A mutant. The cAMP-ligated S128A mutant binds to the consensus DNA binding site with approximately the same affinity as that of cAMP-ligated CRP but forms a different type of complex, which may account for loss of transcriptional activity by the mutant. Energy minimization computations on the cAMP-ligated S128A mutant show that amino acid conformational differences between S128A and CRP occur at Ser179, Glu181, and Thr182 in the center of the DNA binding site, implying that these conformational changes may account for the difference in DNA binding.

[Alkylation of tRNAPhe with 4-(N-2-chloroethyl-N-methylamino) benzyl-5'-phosphamide of d(ATTTTCA)]. / Alkilirovabnie tRNKPhe 4-(N-2-khlorétil-N-metilamino) benzil-5'-fosfamidom d(ATTTTCA).

Alkylation of E. coli tRNAPhe with 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphamide of oligonucleotide d(ATTTTCA) complementary to the sequence UGAAms2i6AA psi in the anticodon loop of tRNAPhe was studied. Three guanine residues--G28/29, G24 and G10 were found to be alkylated. Two binding sites for the reagent in the tRNA were assumed to be present. The efficiency of the alkylation of tRNA from these sites as well as an average association constant (Ka 3,8 X 10(3)M-1) for the reagent interaction with tRNA were evaluated.

[Use of spin-labelled substrate analogs for a comparative study of microsomal cytochrome P-450 and P-448]. / Ispol'zovanie spin-mechenykh analogov substratov dlia sravnitel'nogo izucheniia mikrosomal'nykh tsitokhromov P-450 i P-448.

The previously described, iodine-labeled alkylating stable nitroxyl radicals located at different distances between the N-O. group and the iodine atom were used for a comparative study of the structure of microsomal cytochromes P-450 and P-448 active centers. The radicals were shown to change the optical spectra of Fe3+ located in the active site of the enzyme that are similar to those induced by cytochrome P-450 substrates. Some differences in the type of the radicals binding to control, phenobarbital- and 3-methylcholanthrene-induced microsomes were revealed. The alkylating radical substrate analogs covalently bound to microsomal cytochrome P-450 in the vicinity of the active center, resulting in the inhibition of oxidation of type I and II substrates (e. g., aniline and naphthalene). The value of the spectral binding constant (Ks) for naphthalene in the presence of the radical covalently bound to the cytochrome P-450 active center showed a tendency to increase. Using the ESR technique, the interaction between Fe3+ and the radical localized in the active site of cytochrome P-450 was demonstrated. The contribution of Fe3+ to the relaxation of the radicals covalently bound to cytochrome P-450 was evaluated from the values of the spin label ESR spectra saturation curves at 77K. The distances between the N-O. group of these radicals and Fe3+ in the enzyme active center for the three types of microsomes were determined. The data obtained point to structural peculiarities of the active center of cytochrome P-450, depending on the microsomal type.

[Selective alkylation of G24 residue in tRNAPhe]. / Selektivnoe alkilirovanie tRNAPhe po ostatku G24.

Alkylation of E. coli tRNAPhe with 4-(N-2-chloroethyl-N-methylamino) benzyl-5'-phosphamide of oligonucleotide d(pAACCA) was studied. G24 residue located near the sequence C17GGDA21 partially complementary to the oligonucleotide moiety of the reagent was shown to be alkylated. Oligonucleotide d(pAACCA) inhibited the alkylation. Association constant of oligonucleotide derivative with tRNAPhe (10(3) M-1) was evaluated from the dependence of the extent of tRNA modification on the concentration of the reagent. The reported method for selective alkylation of tRNA may be used for preparing photoaffinity derivatives of tRNA bearing an arylazidogroups in desired position.

[Adenosine- and ethenoadenosine-5'-trimetaphosphates: the effect of covalent bond formation on the state of the affinity label in the complex with phenylalanyl-tRNA-synthetase]. / Adenozin- i étenoadenozin-5'-trimetafosfaty: vliianie obrazovaniia kovalentnoi sviazi na sostoianie affinnoi metki v komplekse s fenilalanil-tRNK-sintetazoi.

epsilon ATP is a substrate of phenylalanyl-tRNA synthetase and epsilon Ado is a competitive inhibitor of ATP in the reaction of tRNA aminoacylation (Ki = 1.6 mM). The association of phenylalanyl-tRNA synthetase with ATP or Ado results in synergistic binding of phenylalaninol and phenylalanine, respectively. However neither epsilon ATP nor epsilon Ado exhibit synergism. Adenosine- and ethenoadenosine-5'-trimethaphosphates are shown to be similar affinity reagents of phenylalanyl-tRNA synthetase. ATP being covalently bound to the enzyme shows essentially lower synergistic effect in comparison with free ATP. epsilon ATP-label is practically insensitive to the ligands namely ATP, Phe, phenylalaninol and is highly accessible for I- ions. The scheme of behaviour of affinity labels is assumed to be as follows: a) the formation of specific reagent-enzyme complex, b) the covalent attachment of the reagent to the enzyme, c) the covalent binding induced disruption of the specific complex formed before.

Co-operative effects in affinity labeling reveal the interaction of tRNA-recognition centers of phenylalanyl-tRNA synthetase.

A mathematical treatment of affinity labeling of the enzymes is presented. The model considered involves a dimeric enzyme with identical ligand binding sites. Equations are derived which describe the kinetics of modification; mutual influence of ligand molecules on association, on the rate of covalent attachment and the possibility of the existence of different sites of modification are taken into account. Experimental data on affinity labeling of phenylalanyl-tRNA synthetase (L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20) of Escherichia coli MRE-600 with N-bromoacetyl-[14C]phenylalanyl-tRNA are treated in terms of the model suggested. The affinity (association constant value) of the tRNAPhe analog molecule towards the enzyme is only slightly affected by another molecule, whereas the reaction rate constant of covalent attachment decreases significantly. The latter is assumed to be due to acceptor site change in the complex containing two molecules of the tRNAPhe analog.

[Interactions of microsomal cytochrome P-450 with spin-labeled substrate analogs]. / Vzaimodeistvie mikrosomal'nogo tsitokhroma P-450 so spin-mechenymi analogami substratov.

The iodine-containing stable iminoxyl radicals with various distances between the N-O-group and the iodine atom are proposed to be used to study the structure of the active center of the microsomal cytochrome P-450. The radicals used induce changes in the optical spectra of the Fe3+ ion located in the active center of the enzyme, as in the case of type 1 substrates and inhibit essentially the microsomal oxidation of cytochrome P-450 substrates of type 1 and 2. This inhibition is neither due to suppression of the NADPH-cytochrome c reductase activity nor to cytochrome P-450 conversion to cytochrome P-420. Cytochrome P-450 substrates (aminopyrine) protect the enzyme against the radical-induced inactivation. The iodine-containing radicals are covalently bound to cytochrome P-450 in the vicinity of active center. The values of dissociation constants for the reversible enzyme-radical constants and the rate constants for the monomolecular transformation in the complex, k, were determined. The EPR method was used to detect the coupling between Fe3+ and the radical located in the active center of cytochrome P-450. The saturation curves of radical SPR spectra at 77 degrees K were employed to determine the contribution of Fe3+ to the relaxation time, T1, of the radicals covalently bound to cytochrome P-450 and to estimate the distances between the Fe3+ ion and the N-O-group of these radicals in the enzyme active center.

[Interaction between tRNA-recognizing sites of phenylalanyl-tRNA synthetase from Escherichia coli MRE-600]. / Vzaimodeistvie tRNK-uznaiushchikh uchastkov v fenilalanil-tRNK-sintetaze iz Escherichia coli MRE-600.

Modification of phenylalanyl-tRNA synthetase by N-Br-Ac-[14C]Phe-tRNAPhe results in two products, one being stable in alkaline conditions and the other being instable. The instable product is formed in the presence of an equimolar excess of the reagent over the enzyme while the stable one--in the presence of a tenfold excess. The experimental results are described by a kinetic model which takes into account the existence of two routes of modification of product formation. The first route is from the complex containing one molecule of the reagent and the other from the complex containing two molecules of the reagent. It is assumed that the presence of the molecule of the reagent in one center of the dimeric enzyme induces a change in the acceptor point of the other center, which leads to local conformational changes of the enzyme molecule interacting with the substrate analogue.

[Exposure of cooperativity of the active sites of rabbit skeletal muscle creatine kinase during its interaction with gamma-amides of ATP]. / Proiavlenie kooperativnosti pri vzaimodeistvii kreatinkinazy iz skeletnykh myshts krolika s gamma-amidami-ATP.

The cooperativity of the active sites of creatine kinase from rabbit skeletal muscle (2.7.3.2) during its interaction with gamma-amides of ATP, e. g. gamma-anilide ATP (I), gamma-benzylamide ATP (II), N-2-hydroxyethyl-N-methyl-gamma-amide ATP (III) and 4-(N-2-hydroxyethyl-N-methylamino)-benzyl-gamma-amide ATP (IV) was studied. A fluorimetric assay of the enzyme complex formation with the analogs was carried out. The values of the intrinsic dissociation constants for the complexes were calculated from the equation for consecutive order of ligands addition. The negative cooperativity of the nucleotide binding sites was found for analogs I and II. Analogs III and IV revealed no noticeable differences in the values of intrinsic dissociation constants. Creatine was shown to have no influence on the cooperativity of the nucleotide binding sites.

[Study of affinity modification kinetics as an approach to demonstrating cooperativity between substrate-recognition centers of bicentric enzymes]. / Izuchenie kinetiki affinnoi modifikatsii kak podkhod k vyiavleniiu kooperativnosti mezhdu tsentrami uznavaniia substratov dvukhtsentrovykh fermentov.

The dependence of the initial rate of the affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 by an phenylalanyl-tRNA analog--N-Br-Ac-[14C]Phe-tRNAPhe against reagent concentration was obtained. The curve runs through maximum therefore it is impossible to describe it by the traditional Kitz and Wilson scheme. The experimental results were treated in assumption of dimeric enzyme, cooperativity in the substrate analog binding and its conversion being taken into account. It was shown that such a treatment of kinetic data of the affinity labelling allows to estimate quantitatively the cooperativity arisen between substrate analog centers of the binding and conversion. The data obtained allow to assume that in the case of phenylalanyl-tRNA analog there is a slight negative cooperativity in the reagent binding (thermodynamic cooperativity) but strong negative cooperativity in reagent conversion (kinetic cooperativity). The results obtained testify the high sensitivity of the kinetic approach for elucidation of centers cooperativity.

[Phenylalanyl-tRNA synthetase from E. coli MRE-600. Effect of chemical modification of lysine residues on the enzyme interaction with substrates]. / Fenilalanil-tRNK-sintetaza iz E. coli MRE-600. Vliianie khimicheskoi modifikatsii ostatkov lizina na vzaimodeistvie fermenta s substratami.

The effect of modification of Phe-RSase from E. coli MRE-600 by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP and L-phenylalanynyl-5'-adenylate obtained by periodate oxidation on the enzyme interaction with substrates was investigated. It was shown that modification of Phe-RSase by pyridoxal-5'-phosphate and 2', 3'-dialdehyde derivative of ATP leads to a decrease of the aminoacylation rate without changing the rate of the ATP-[32P]-pyrophosphate exchange reaction. The substrate analogs L-phenylalanynol and L-phenyl-alanynyladenylate increase the degree of Phe-RSase inactivation in the aminoacylation reaction. tRNAphe strongly protects the enzyme against inactivation. ATP, both in the absence (in case of modification with pyridoxal-5'-phosphate) and in- the presence of Mg2+ and phenylalanine (in case of modification with o-ATP) exhibits a pronounced protective effect. L-Phe does not protect the enzyme against the inactivation by pyridoxal-5'-phosphate or o-ATP. The dissociation constant of the Phe-RSase[14C]-Phe-tRNAphe complex increases 2.5 -- 5-fold after the enzyme modification by pyridoxal-5'-phosphate, while the Km value for tRNAphe decreases approximately two times in the aminoacylation reaction. There are no changes in the Km values for amino acid and ATP and the Hill coefficients for all substrates tested. Modification of Phe-RSase by pyridoxal-5'-phosphate leads to a decrease of stability of the aminoacyladenylate -- enzyme complex. Oxidized L-phenylalanynyladenylate does not produce enzyme inactivation either by aminoacylation or in the isotropic ATP-PP iota exchange reaction. It is assumed that Phe-RSase from E. coli MRE-600 contains some lysine residues essential for binding and aminoacylation of tRNA, which do not occur in the ATP-binding subsite and aminoacyladenylate formation center.

[Role of the carboxylic groups in interaction of phenylalanyl-tRNA synthetase with substrates]. / Rol' karboksil'nykh grupp vo veaimodeistvii fenilalanil-tRNK-sintetazy s substratami.

The effect of modification of carboxylic groups of phenylalanyl-tRNA synthetase by p-toluene sulfonate N-cyclohexyl-N'-beta-(4-methylmorpholine) ethylcarbodiimide (CMEC) on the activity of the enzyme was investigated. It was shown that modification of two moles of carboxylic groups per mole of the enzymes leads to the diminution of negative charge of the enzyme and to inactivation in ATP-[32P]PPi-exchange and aminoacylation reactions. The inactivation is completely reversed by mild alkaline hydrolysis. ATP in concentration 2 X 10(-4) M partially protects the enzyme against inactivation, protective effect being stimulated by Mg2+ and 0.4-0.7 moles of carboxylic groups per mole of the enzyme are protected against inactivation is observed although the depth of modification is increased. Other substrates do not have protective effect. Modification of the enzyme by CMEC increases Kdiss value of [14C]-Phe-tRNA enzyme complex and Km value for tRNAPhe in aminoacylation by factor of three. Vmax for all substrates in both aminoacylation and leads to 40% increase of Hill's coefficient for ATP in ATP-[32P]PPi-exchange reaction but not in aminoacylation. The carboxylic groups modified by CMEC are assumed to take part in ATP recognition and in catalysis of the ATP conversion and in catalysis of transfer of activated amino acid residues on tRNA.

[Reaction between rabbit skeletal muscle creatine kinase and ATP gamma-amides]. / Vzaimodeistvie kreatinkinazy iz skeletnykh myshts krolika s gamma-amidami ATP.

The interaction of creatine kinase from skeletal muscles of rabbit with ADP, ATP and gamma-amides of ATP: gamma-anilide ATP (1), gamma-benzylamide ATP (2), N-2-hydroxyethyl-N-methyl-gamma-amide ATP (3) and 4-(N-2-hydroxyethyl-N-methylamino)-benzyl-gamma-amide ATP (4) was investigated. The values of dissociation constants of nucleotides were determined by fluorescence quenching method. All analogs preserve their affinity to the enzyme, analogs 1 and 2 being the strongest in affinity. The values of dissociation constants of these analogs are equal to those for ATP and ADP. The influence of Mg2+ and creatine on gamma-amides ATP enzyme binding was investigated. The affinity of gamma-amides of ATP in the presence of Mg2+ and creatine was shown to decrease. It is concluded that gamma-amides of ATP (1,2) have the suitable structure for the preparation of affinity reagents for creatine kinase.

[Role of arginine residues in phenylalanyl-tRNA synthetase interaction with substrates]. / Rol' ostatkov arginina vo vzaimodeistvii fenilalanil-tRNK-sintetazy s substratami.

The effect of 2,4-pentandione on the activity of phenylalanyl-tRNA synthetase (Phe-RSase) from E. coli MRE-600 was investigated. Modification of arginine residues of Phe-RSase with 2,4-pentandione was shown to decrease the rate of both ATP-[32P]pyrophosphate exchange and aminoacylation reaction. In the presence of Mg2+ ions ATP essentially protects the enzyme from inactivation. L-Phe has practically no protective effect. There are no changes in the values of Km for tRNAPhe in the aminoacylation reaction and for amino acid in ATP-[32P]pyrophosphate exchange and aminoacylation reaction while Km for ATP is increased in both. Modification of 42-44 arginine residues per molecule of the enzyme causes loss of 50% of the enzyme activity for tRNA aminoacylation. Under these conditions ATP in the presence of Mg2+ ions as well as ATP + Mg2+ combined with L-Phe, protects 8-10 arginine residues per molecule of the enzyme from modification. Part of arginine residues of the enzyme molecule is assumed to be essential for ATP binding.

[Modification of one tRNA recognition site of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA]. / Blokirovanie tRNK-uznaiushchego uchastka fenilalanil-tRNK-sintetazy iz Escherichia coli MRE-600 S pomoshch'iu N-khlorambutsilil-fenilalanil-tRNK.

Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA results in a binding of 1 mole of the reagent per 1 mole of the enzyme. Exhaustive alkylation of phenylalanyl-tRNA synthetase completely blocks the aminoacylation and partially inhibits the reaction of ATP--[32P]pyrophosphate exchange. Removal of the tRNA moiety of the reagent by hydrolysis of the ester bond N-chlorambucilyl-phenylalanine and terminal adenosine does not result in a restoration of ATP--[32P]pyrophosphate exchange and aminoacylation activity. The latter result may testify a chemical modification of amino acid residues essential for enzymatic activity. Possibility of blocking one of the two tRNA binding sites is discussed.

[Role of the lysine residues in the phenylalanyl-tRNA synthetase substrates interaction]. / Rol' ostatkov lizina vo vzaimodeistvii fenilalanil-tRNK-sintetazy s substratami.

The effect of 2,4-pentandione on the activity of phenylalanyl-tRNA synthetase (Phe-RSase) from E. coli MRE-600 was investigated. It was shown that modification of Phe-RSase with 2,4-pentandione leads to decrease of the aminoacylation rate without an influence on the ATP--[32P]pyrophosphate exchange reaction rate. tRNAPhe protects the enzyme against inactivation. Neither L-Phe and ATP nor the analog fo aminoacyladenylate protects the enzyme against inactivation. There are no changes in Km for amino acid and ATP in the aminoacylation reaction after modification while Km for tRNAPhe decreases three times. The dissociation constant of Phe-RSase: [14C]Phe-tRNA complex increases 4--8 times after modification. It is assumed that there are some lysine residues in Phe-RSase essential for the Phe-RSase-tRNA interaction.

Affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 by E. coli tRNAphe containing photoreactive group.

The photoinduced reaction of phenylalanyl-tRNA synthetase (E.C.6.1.1.20) from E.coli MRE-600 with tRNAphe containing photoreative p-N3-C6H4-NHCOCH2-group attached to 4-thiouridine sU8 (azido-tRNAphe) was investigated. The attachment of this group does not influence the dissociation constant of the complex of Phe-tRNAphe with the enzyme, however it results in sevenfold increase of Km in the enzymatic aminoacylation of tRNAphe. Under irradiation at 300 nm at pH 5.8 the covalent binding of [14C]-Phe-azido-tRNAphe to the enzyme takes place 0.3 moles of the reagent being attached per mole of the enzyme. tRNA prevents the reaction. Phenylalanine, ATP,ADP,AMP, adenosine and pyrophosphate (2.5 xx 10(-3) M) don't affect neither the stability of the tRNA-enzyme complex nor the rate of the affinity labelling. The presence of the mixture of either phenylalanine or phenylalaninol with ATP as well as phenylalaninol adenylate exhibits 50% inhibition of the photoinduced reaction. Therefore, the reaction of [14C]-Phe-azido-tRNA with the enzyme is significantly less sensitive to the presence of the ligands than the reaction of chlorambucilyl-tRNA with the reactive group attached to the acceptor end of the tRNA studied in 1. It has been concluded that the kinetics of the affinity labelling does permit to discriminate the influence of the low molecular weight ligands of the enzyme on the different sites of the tRNA enzyme interaction.