S. N. Mikhailov

Disaccharide Nucleosides and Oligonucleotides on Their Basis. New Tools for the Study of Enzymes of Nucleic Acid Metabolism

The main structural features of an important group of natural compounds, disaccharide nucleosides, are reviewed. The synthesis and properties of modified oligonucleotides on their basis as well as the methods of introduction of reactive aldehyde groups are described. The last part is devoted to the application of these compounds for studies of enzymes of nucleic acid metabolism.

Substrate specificity of Escherichia coli thymidine phosphorylase

Substrate specificity of Escherichia coli thymidine phosphorylase to thymidine derivatives modified at 5′-, 3′-, and 2′,3′-positions of the sugar moiety was studied. Equilibrium and kinetic constants (Km, KI, kcat) of the phosphorolysis reaction have been determined for 20 thymidine analogs. The results are compared with X-ray and molecular dynamics data. The most important hydrogen bonds in the enzyme-substrate complex are revealed.

Substrate properties of C' -methyl UTP derivatives in T7 RNA polymerase reactions. Evidence for N-type NTP conformation

The number of synthetic UTP analogues containing methyl groups in different positions of the ribose moiety were tested as substrates for T7 RNA polymerase (T7 RNAP). Two of these compounds (containing substituents in the 5′ position) were shown to be weak substrates of T7 RNAP. 3′Me‐UTP was neither substrate nor inhibitor of T7 RNAP while 2′Me‐UTP was shown to terminate RNA chain synthesis. Conformational analysis of the analogues and parent nucleotide using the force‐field method indicates that the allowed conformation of UTP during its incorporation into the growing RNA chain by T7 RNAP is limited to the χ angle range of 192–256° of N‐type conformation.

Determination of the nucleotide conformation in the productive enzyme-substrate complexes of RNA-depolymerases

© 1997 Federation of European Biochemical Societies.

A route to 2',5'-oligoadenylates with increased stability towards phosphodiesterases.

The rates of enzymatic hydrolysis of 2′,5′‐oligoadenylates and their synthetic analogs have been measured. These compounds were treated with either NIH 3T3 cell lysates, mouse liver homogenates or snake venom phosphodiesterase. All analogs with 3′‐terminal acyclic nucleoside residues demonstrated greater stability compared with the natural compound adenylyl(2′‐5′)adenylyl(2′‐5′)adenosine.

Use of 4-Thiouridine and 4-Thiothymidine in Studies on Pyrimidine Nucleoside Phosphorylases

The new substrates 4-thiouridine and 4-thiothymidine were proposed for spectrophotometric measurement of the activity of uridine (UP) and thymidine (TP) phosphorylases. At pH 7.5, 4-thiouridine has an absorbance maximum at 330 nm, and the difference in extinction coefficient (Δε) between 4-thiouridine and 4-thiouracil is 3000 М–1cm–1. 4-Thiouridine proved to be a good substrate for UP: the Michaelis (КМ) and catalytic (kcat) constants were estimated respectively at 130 μM and 49 s–1 at 25°C. Even a greater Δε (5000 M–1cm–1 at 336 nm) was observed for the 4-thiothymidine/4-thiothymine pair.

Interaction of HIV-1 Reverse Transcriptase and T7 RNA Polymerase with Phosphonate Analogs of NTP and Inorganic Pyrophosphate

We have examined the interaction of human immunodeficiency virus reverse transcriptase (HIV RT) and T7 RNA polymerase (T7 RNAP) with modified nucleoside triphosphates and inorganic pyrophosphate (PPi) analogs containing nonhydrolyzable bisphosphonate groups. We have synthesized a number of derivatives of bisphosphonic acid having different aromatic and nonaromatic side substituents, as well as the NTP derivatives whose incorporation into the growing nucleotide chain during the polymerization reaction results in formation of bisphosphonates as leaving groups. The competitive character of inhibition of both enzymes has been revealed for all the compounds under study, and the inhibition constants have been estimated. One of PPianalogs containing a bulky aromatic substituent is characterized by similar inhibition constants for both T7 RNAP and RT. The universal character of this inhibitor can serve as evidence for a similar structure of the NPT-binding sites in the two polymerases. It has been shown that nonsubstituted methylenebisphosphate is a better leaving group than that containing additional methyl and hydroxyl groups. The NTP analogs are very weak inhibitors of T7 RNAP, whereas HIV RT is more sensitive to this type of compounds. On the basis of the X-ray crystallographic data on the T7 RNAP complex with a template and NTP, we have modeled the binding of some derivatives of bisphosphonic acid in the active center of the enzyme. The peculiarities observed in the model correlate well with the experimental data on inhibition.

Synthesis and Properties of Novel NTP Derivatives

Abstract Simple method for the preparation of anhydrides of nucleoside-5′-monophosphoric acid and with 1-hydroxyethane-1,1-diylbis(phosphonic acid) has been developed.

Probing the MvaI Methyltransferase Region that Interacts with DNA: Affinity Labeling with the Dialdehyde-Containing DNA Duplexes

Abstract Affinity labeling of methyltransferase MvaI by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine or 1-(β-D-galactopyranosyl)thymine residues was performed. Partial chemical hydrolysis of the covalently bound methylase in the conjugates with the dialdehyde-containing DNA allowed us to determine the amino acid region in the C terminus of methylase MvaI that interacts with DNA.

[Modified oligonucleotides containing 1-beta-D-galactopyranosylthymine: synthesis and substrate properties].

A convenient method of regioselective introduction of 1-β-D-galactopyranosylthymine into oligonucleotides was developed and the substrate properties of the modified oligonucleotides were investigated in the enzymatic reactions of formation and hydrolysis of internucleotide bonds.