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Biomedical subjects

S N Kochetkov

Publications and source records attributed to S N Kochetkov.

At least 37 records · Page 2Linked to original sources
PubMed1995

Targeted mutagenesis identifies Asp-569 as a catalytically critical residue in T7 RNA polymerase.

In order to look more closely at a well-conserved region in T7 RNA polymerase (T7 RNAP) containing, as shown earlier, the functionally essential residues Pro-563 and Tyr-571, we used targeted mutagenesis to change those residues within this region that are invariant in all single-subunit RNA polymerases, and characterized the mutant enzymes in vitro. The most interesting finding of this study was the crucial importance of the acidic group of Asp-569. In addition, we have shown that the phenolic ring is the most significant functional group of Tyr-571, with the hydroxy group also contributing to promoter binding.

Amino Acid Sequence↗
PubMed1994

The studies of cooperative regions in T7 RNA polymerase.

The heat denaturation of bacteriophage T7 RNA polymerase (T7RNAP) was studied by scanning microcalorimetry. The thermodynamic parameters of the denaturation were estimated within the pH range 6-9. The analysis of the denaturation curves showed the presence of two cooperative parts of the T7RNAP molecule melting according to the 'all-or-none' principle. The molecular masses of these parts were determined as 22 and 77 kDa. These values are close to the molecular masses of protein domains obtained from X-ray diffraction and limited trypsinolysis data. The smaller N-terminal domain was shown to increase the thermostability of the 'catalytic' C-terminal domain within the intact T7RNAP molecule.

Bacteriophage T7↗
PubMed1994

[Studies of the resistance of the human immunodeficiency virus to azidothymidine. I. Kinetic studies of the interaction between reverse transcriptase and a series of its mutant forms with substrates and azidothymidine-5'-trisphosphate].

Prolonged therapy of AIDS patients with azidothymidine results in the development of resistance to the drug. This phenomenon is accompanied by the appearance of point mutations in the pol gene coding for reverse transcriptase (RT). Kinetic studies of interactions of wild type RT and its forms containing the above-mentioned mutations with substrates and azidothymidine 5'-triphosphate have been carried out. Considerable differences in the affinities of RT and the mutants for RNA and DNA heteropolymer templates were established. The mutations did not affect the RT affinity for dNTP; however, its location influenced considerably the inhibition of the reaction with azidothymidine 5'-triphosphate.

Amino Acid Sequence↗
PubMed1994

[Functional studies of mutant forms of bacteriophage T7 RNA polymerases containing point substitutions in the motif at the active site of the enzyme].

Monomeric DNA and RNA polymerases contain a number of conserved motifs. By means of random and oligonucleotide-directed mutagenesis the point mutants of bacteriophage T7 RNA polymerase containing the amino acid substitutions in motif B which is the part of the active site were obtained. The kinetic properties of the mutants were studied. The results obtained suggest that amino acid residues 636, 639 and 646 are involved in the binding of the initiating GTP, the selection of correct nucleoside triphosphate and interaction with the promoter, respectively.

Amino Acid Sequence↗
PubMed1993

Interactions of the HIV-1 reverse transcriptase 'AZT-resistant' mutant with substrates and AZT-TP.

To investigate the biochemical basis of the HIV-1 resistance to AZT we obtained the RT mutant containing four amino acid substitutions by an oligonucleotide-directed mutagenesis technique. Enzymatic properties of the wild type and mutant RTs were compared. 'AZT-resistant' mutations in RT were shown to be associated with the reduced capability of AZT-TP to block the DNA- but not RNA-directed DNA synthesis.

Antiviral Agents↗
PubMed1993

Tyr-571 is involved in the T7 RNA polymerase binding to its promoter.

The in vitro studies of three T7 RNA polymerase point mutants suggest that substitutions of Ala and Thr for Pro-563 and of Ser for Tyr-571 have little effect on the enzyme catalytic competence, but result in its inability to utilize the promoter. Both P563A and P563T mutants retain the promoter-binding ability, whereas the promoter affinity of the Y571S mutant drops drastically.

Amino Acid Sequence↗
PubMed1993

Random mutagenesis of the gene for bacteriophage T7 RNA polymerase.

Random mutagenesis of the gene for bacteriophage T7 RNA polymerase was used to identify functionally essential amino acid residues of the enzyme. A two-plasmid system was developed that permits the straightforward isolation of T7 RNA polymerase mutants that had lost almost all catalytic activity. It was shown that substitutions of Thr and Ala for Pro at the position 563, Ser for Tyr571, Pro for Thr636, Asp for Tyr639 and of Cys for Phe646 resulted in inactivation of the enzyme. It is noteworthy that all these mutations are limited to two short regions that are highly conservative in sequences of monomeric RNA polymerases.

Amino Acid Sequence↗
PubMed1993

[Study of phage T7 DNA-dependent RNA-polymerase using GTP analogs. Affinity modification and study of interaction with matrices using fluorescent markers].

Interactions of the bacteriophage T7 DNA-dependent RNA polymerase with three GTP analogs have been studied. All of the three analogs tested contained substituted naphthalenesulphamide groups and were shown to be under appropriate conditions irreversible covalent inhibitors of the enzyme, the modified enzyme possessing fluorescent properties. One of these analogs contained the reactive 2-bromoethyl phosphonate group and was shown to cause the loss of the enzyme affinity for polynucleotide templates. The other two modifiers which contained the azide reactive group did not alter the enzyme-template affinity, the polynucleotide binding leading to a notable increase of the enzyme fluorescence intensity. The latter two modifiers are supposed to be convenient for fluorescent labelling of the active site of RNA polymerase for enzyme-template binding studies.

Affinity Labels↗
PubMed1992

On the functional role of the Tyr-639 residue of bacteriophage T7 RNA polymerase.

Substitution of Asp for a Tyr residue normally present at position 639 of the bacteriophage T7 RNA polymerase leads to a drastic drop in the enzymatic activity. This mutation does not affect the enzyme-promoter interaction but decreases the ability of the RNA polymerase to discriminate between GTP and ATP molecules, resulting in a decrease in the rate of the incorporation of the nucleotide into the RNA chain.

Amino Acid Sequence↗
PubMed1991

Lys631 residue in the active site of the bacteriophage T7 RNA polymerase. Affinity labeling and site-directed mutagenesis.

A highly selective affinity labeling of T7 RNA polymerase with the o-formylphenyl ester of GMP and [alpha-32P]UTP was carried out. The site of the labeling was located using limited cleavages with hydroxylamine, bromine, N-chlorosuccinimide and cyanogene bromide and was identified as the Lys631 residue. Site-directed mutagenesis using synthetic oligonucleotides was used to substitute Lys631 by a Gly, Leu or Arg residue. Kinetic studies of the purified mutant enzymes showed alterations of their polymerizing activity. For the Lys----Gly mutant enzyme, anomalous template binding was observed.

Affinity Labels↗
PubMed1990

Inactivation of bacteriophage T7 DNA-dependent RNA polymerase by 5'-p-fluorosulfonylbenzoyladenosine. Identification of the modification site and the effect of the modification on enzyme action.

Bacteriophage T7 RNA polymerase was covalently modified by 5'-[4-fluorosulfonyl)benzoyl]adenosine (4-FSO2BzAdo). The modified enzyme lacks the ability to catalyze RNA synthesis from the phi 10 promoter of bacteriophage T7; both promoter and GTP binding being markedly decreased. The mild hydrolysis of the ester bond of 4-FSO2BzAdo within the covalent enzyme-inhibitor complex restores the RNA synthesis at a lower rate. Sequence studies show that Lys172 is the target of modification by 4-FSO2BzAdo. This residue, which is situated in the polypeptide region connecting two domains of RNA polymerase, was shown to be the primary site of the limited proteolysis occurring in vivo [Ikeda, R. A. & Richardson, C. C. (1987) J. Biol. Chem. 262, 3790-3799]. We propose that Lys172 is located outside the active site. Once this residue has reacted with 4-FSO2BzAdo, the nucleoside moiety of the analog is fixed in the NTP-binding site of the active centre and prevents binding of the substrates. Here, Lys172 per se is not important for the activity but serves as an 'anchor' for binding of the inhibitor.

Adenosine↗
PubMed1990

T7 RNA polymerase: use of limited proteolysis for the study of enzyme's interaction with substrates and inhibitors.

The specific limited trypsinolysis of bacteriophage T7 RNA polymerase (T7RP) was performed in the presence of various components of the polymerase reaction and some GTP-analogs--irreversible inhibitors of the enzyme. The differences in the rate and sites of proteolysis were observed. Basing on the data obtained the role of the N-terminal domain of the T7RP in the interaction with promoter-containing template is proposed.

DNA↗
PubMed1990

[The study of nucleoside triphosphate-binding center of DNA-dependent RNA-polymerase of phage T7 using GTP analogs].

The NTP binding site of bacteriophage T7 DNA-dependent RNA polymerase was studied using GTP analogs. For four analogs the irreversible inhibition was demonstrated. The kinetic parameters for competitive (Ki) and irreversible (KI and k3) inhibition were determined. One of the analogs, 5'[2-hydroxy(4-iodoacetamido)benzoyl]guanosine, was shown to inactivate the enzyme rapidly due to the modification of SH-groups. Some suggestions on the structure of the RNA polymerase active site have been made.

Binding Sites↗
PubMed1987

[Mechanism of activation of cAMP-dependent protein kinase. Covalent modification of the holoenzyme by cupric 1,10-phenanthroline].

Modification of the cAMP-dependent protein kinase holoenzyme from porcine brain by cupric-1,10-phenanthroline was studied. The formation of conjugates with Mr of 180,000 and 100,000 Da, whose stoichiometry coincides with that of holoenzymes R2C2 and RC, and of a conjugate with Mr of 85,000 Da corresponding to the catalytic subunit conjugate with the proteolytic fragment of the regulatory subunit (Mr = 39,000 Da) was demonstrated. The modified enzyme was activated by cAMP.

Animals↗
PubMed1987

[A thermostable protein inhibitor of protein kinase from the swine brain. Isolation of the inhibitor and interaction with the enzyme].

A heat-stable protein inhibitor of cAMP-dependent protein kinase has been isolated from pig brain tissue. During gel filtration the protein is eluted in three peaks corresponding to the tetramer, dimer and monomer. The monomer fraction was purified 609-fold. The molecular mass of the monomeric form as determined by gel filtration and electrophoresis is equal to 11,000 Da and 8000 Da, respectively. The inhibition of the phosphotransferase reaction with respect to ATP occurs via a non-competitive mechanism, while that for histone--via a competitive mechanism. A formal kinetic analysis of various modes of the inhibitor binding to different protein kinase forms, e. g., the cAMP-dependent protein kinase catalytic subunit, protein kinase holoenzyme in the presence and absence of cAMP as well as of holoenzyme preparations modified by dimethylsuberimidate and cupric 1,10-phenanthroline, has been carried out. It was demonstrated that 3-4 inhibitor molecules are involved in the interaction with protein kinase.

Amino Acids↗