Human immunodeficiency virus reverse transcriptase: steady-state and pre-steady-state kinetics of nucleotide incorporation

Steady-state and pre-steady-state kinetic constants were determined for reverse transcriptase catalyzed incorporation of nucleotides and nucleotide analogues into defined-sequence DNA primed-RNA templates. 3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) was almost as efficient a su...

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Vydáno v:Biochemistry (Easton) Ročník 31; číslo 18; s. 4473
Hlavní autor: Reardon, J E
Médium: Journal Article
Jazyk:angličtina
Vydáno: United States 12.05.1992
Témata:
Antiviral Agents - pharmacology
Base Sequence
Binding, Competitive
Dideoxynucleotides
HIV - enzymology
Molecular Sequence Data
Oligonucleotide Probes - chemistry
Reverse Transcriptase Inhibitors
RNA-Directed DNA Polymerase - chemistry
Templates, Genetic
Thionucleotides - chemistry
Thymine Nucleotides - pharmacokinetics
Thymine Nucleotides - pharmacology
Zidovudine - analogs & derivatives
Zidovudine - pharmacology
ISSN:0006-2960
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Shrnutí:Steady-state and pre-steady-state kinetic constants were determined for reverse transcriptase catalyzed incorporation of nucleotides and nucleotide analogues into defined-sequence DNA primed-RNA templates. 3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) was almost as efficient a substrate (kcat/Km) as dTTP for the enzyme. In contrast, the four 2',3'-dideoxynucleoside 5'-triphosphates and 3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (d4TTP) were 6-30-fold less efficient substrates of the enzyme. The kcat values for all nucleotide analogues were similar, consistent with a kinetic model in which the steady-state rate-limiting step was dissociation of the template-primer from the enzyme [Reardon, J. E., & Miller, W. H. (1990) J. Biol. Chem. 265, 20302-20307]. The pre-steady-state kinetics of single-nucleotide incorporation were consistent with the kinetic model: [formula: see text] where E, TP, and dNTP represent reverse transcriptase, a defined-sequence DNA primed-RNA template, and 2'-deoxynucleoside 5'-triphosphate (or analogue), respectively. The dissociation constant (Kd1) for template-primer binding was 10 nM, and the estimated rate constants for association and dissociation of the enzyme.template-primer complex were 4 x 10(6) M-1 s-1 and 0.04 s-1, respectively. The dissociation constants (Kd2) for dTTP, AZTTP, and 3'-deoxythymidine 5'-triphosphate (ddTTP) were 9, 11, and 4.6 microM, respectively. Thus, the differences in steady-state Km values were not due to differences in binding of the nucleotide analogues to the enzyme. In contrast, the rate-limiting step during single-nucleotide incorporation (kp) was sensitive to the structure of the nucleotide substrate.
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ISSN:0006-2960
DOI:10.1021/bi00133a013