ABSTRACT
A thymidine kinase UL23 gene (EC 2.7.1.145) from an acyclovir-sensitive strain L2 of herpes simplex virus type 1 was cloned and expressed in E. coli. Enzyme was purified by chromatography to a homogeneous state controlled by PAG electrophoresis. The Michaelis constants for the reactions with thymidine and an acyclovir were determined. It was found that enzyme phosphorilate some modified nucleosides such as d2T, d4T, d2C, 3TC, FLT, BVDU, GCV. A comparison of the purified enzyme properties and properties ofthymidine kinase of other strains of herpes simplex virus, previously published was carried out. It is shown that enzyme is inhibited by acyclovir H-phosphonate.
Subject(s)
Acyclovir/pharmacology , Simplexvirus/enzymology , Thymidine Kinase/metabolism , Amino Acid Sequence , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Cloning, Molecular , Escherichia coli/genetics , Gene Expression , Humans , Molecular Sequence Data , Mutation , Sequence Analysis, DNA , Thymidine Kinase/antagonists & inhibitors , Thymidine Kinase/chemistry , Thymidine Kinase/isolation & purificationABSTRACT
Bacteriophage T5 deoxynucleoside monophosphate kinase (dNMP kinase, EC 2.7.4.13) is shown to catalyze the phosphorylation of both d(2)CMP and ribonucleotides AMP, GMP, and CMP, but does not phosphorylate UMP. For natural acceptors of the phosphoryl group, K(m) and k(cat) were found. The applicability of T5 dNMP kinase as a universal enzyme capable of the phosphorylation of labelled r/dNMP was shown for the synthesis of [alpha-(32)P]rNTP and [alpha-(32)P]dNTP.
Subject(s)
Nucleotides/chemistry , Phosphotransferases (Phosphate Group Acceptor)/chemistry , Siphoviridae/enzymology , Viral Proteins/chemistry , Isotope Labeling/methods , Phosphorus Isotopes/chemistry , Substrate SpecificityABSTRACT
Isosteric triphosphonate derivatives of 2',3'-dideoxy-2',3'-didehydroadenosine and 3'-deoxy-2',3'-didehydrothymidine and their beta,gamma-substituted analogues were synthesized. Their substrate properties toward a number of reverse transcriptases of the human immunodeficiency and bird myeloblastosis viruses, human DNA polymerases alpha and beta, and the Klenow fragment of Escherichia coli DNA polymerase I were studied.
Subject(s)
Adenosine Monophosphate/analogs & derivatives , DNA-Directed DNA Polymerase/chemistry , Deoxyadenine Nucleotides/chemistry , Dideoxynucleotides/chemistry , Organophosphonates/chemistry , Stavudine/analogs & derivatives , Adenosine Monophosphate/chemistry , Animals , Dideoxynucleotides/chemical synthesis , Humans , Stavudine/chemistry , Substrate SpecificityABSTRACT
The total fraction of aminoacyl-tRNA synthases from Escherichia coli has been shown to catalyze the synthesis of the bis(5'-nucleosidyl) oligophosphates Ap4AZT, Ap4d4T, Ap43TC, and Ap4ACV, as well as Ap3AZT and Ap3d4T, from [alpha-32P]ATP and the corresponding nucleoside-5'-tri(or di)phosphate. The resulting compounds, characterized by HPLC, are resistant to alkaline phosphatase. Ap4AZT, Ap4d4T, and Ap43TC are formed with approximately equal efficiency, whereas the efficiencies of the synthesis of Ap4ACV, Ap3AZT, and Ap3d4T are three- to fivefold lower. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.
