Human immunodeficiency virus reverse transcriptase. A kinetic analysis of RNA-dependent and DNA-dependent DNA polymerization
A minimal kinetic mechanism for HIV reverse transcriptase (RT)-catalyzed RNA-dependent and DNA-dependent DNA polymerization was determined by pre-steady-state kinetic methods to be: [formula: see text] where E, TP, dNTP, and PPi are RT, template-primer, 2'-deoxynucleoside 5'-triphosphate,...
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| Vydáno v: | The Journal of biological chemistry Ročník 268; číslo 12; s. 8743 |
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| Hlavní autor: | |
| Médium: | Journal Article |
| Jazyk: | angličtina |
| Vydáno: |
United States
25.04.1993
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| ISSN: | 0021-9258 |
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| Abstract | A minimal kinetic mechanism for HIV reverse transcriptase (RT)-catalyzed RNA-dependent and DNA-dependent DNA polymerization was determined by pre-steady-state kinetic methods to be: [formula: see text] where E, TP, dNTP, and PPi are RT, template-primer, 2'-deoxynucleoside 5'-triphosphate, and inorganic pyrophosphate, respectively. Defined sequence template-primers that encode for incorporation of dTTP were prepared by annealing either a 44-mer RNA template or a 44-mer DNA template (of the same sequence) to a 21-mer DNA primer (r44:d21-mer and d44:d21-mer, respectively). The values of the above kinetic constants were determined for dTMP and 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP) incorporation into both template primers. The kcat and Km values calculated from these kinetic constants were similar to the values directly determined from steady-state experiments. Further, the net rate constants for processive incorporation of three successive nucleotides into the r44:d21-mer were similar indicating that a rate-determining step did not follow catalysis. A 20-fold difference in the rate constants (kp) for incorporation of dTMP into the r44:d21-mer versus the d44:d21-mer was largely responsible for the difference in the calculated processivity numbers of 340 and 5, respectively. Finally, the rate constant for pyrophosphorolysis of the 3'-AZTMP-terminated r44:d21-mer (kpyro) was similar to the rate constant for dissociation of the chain-terminated template primer from the enzyme (koff) indicating that millimolar concentrations of intracellular inorganic pyrophosphate would be required for pyrophosphorolysis of AZTMP-terminated retroviral genomes. |
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| AbstractList | A minimal kinetic mechanism for HIV reverse transcriptase (RT)-catalyzed RNA-dependent and DNA-dependent DNA polymerization was determined by pre-steady-state kinetic methods to be: [formula: see text] where E, TP, dNTP, and PPi are RT, template-primer, 2'-deoxynucleoside 5'-triphosphate, and inorganic pyrophosphate, respectively. Defined sequence template-primers that encode for incorporation of dTTP were prepared by annealing either a 44-mer RNA template or a 44-mer DNA template (of the same sequence) to a 21-mer DNA primer (r44:d21-mer and d44:d21-mer, respectively). The values of the above kinetic constants were determined for dTMP and 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP) incorporation into both template primers. The kcat and Km values calculated from these kinetic constants were similar to the values directly determined from steady-state experiments. Further, the net rate constants for processive incorporation of three successive nucleotides into the r44:d21-mer were similar indicating that a rate-determining step did not follow catalysis. A 20-fold difference in the rate constants (kp) for incorporation of dTMP into the r44:d21-mer versus the d44:d21-mer was largely responsible for the difference in the calculated processivity numbers of 340 and 5, respectively. Finally, the rate constant for pyrophosphorolysis of the 3'-AZTMP-terminated r44:d21-mer (kpyro) was similar to the rate constant for dissociation of the chain-terminated template primer from the enzyme (koff) indicating that millimolar concentrations of intracellular inorganic pyrophosphate would be required for pyrophosphorolysis of AZTMP-terminated retroviral genomes.A minimal kinetic mechanism for HIV reverse transcriptase (RT)-catalyzed RNA-dependent and DNA-dependent DNA polymerization was determined by pre-steady-state kinetic methods to be: [formula: see text] where E, TP, dNTP, and PPi are RT, template-primer, 2'-deoxynucleoside 5'-triphosphate, and inorganic pyrophosphate, respectively. Defined sequence template-primers that encode for incorporation of dTTP were prepared by annealing either a 44-mer RNA template or a 44-mer DNA template (of the same sequence) to a 21-mer DNA primer (r44:d21-mer and d44:d21-mer, respectively). The values of the above kinetic constants were determined for dTMP and 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP) incorporation into both template primers. The kcat and Km values calculated from these kinetic constants were similar to the values directly determined from steady-state experiments. Further, the net rate constants for processive incorporation of three successive nucleotides into the r44:d21-mer were similar indicating that a rate-determining step did not follow catalysis. A 20-fold difference in the rate constants (kp) for incorporation of dTMP into the r44:d21-mer versus the d44:d21-mer was largely responsible for the difference in the calculated processivity numbers of 340 and 5, respectively. Finally, the rate constant for pyrophosphorolysis of the 3'-AZTMP-terminated r44:d21-mer (kpyro) was similar to the rate constant for dissociation of the chain-terminated template primer from the enzyme (koff) indicating that millimolar concentrations of intracellular inorganic pyrophosphate would be required for pyrophosphorolysis of AZTMP-terminated retroviral genomes. A minimal kinetic mechanism for HIV reverse transcriptase (RT)-catalyzed RNA-dependent and DNA-dependent DNA polymerization was determined by pre-steady-state kinetic methods to be: [formula: see text] where E, TP, dNTP, and PPi are RT, template-primer, 2'-deoxynucleoside 5'-triphosphate, and inorganic pyrophosphate, respectively. Defined sequence template-primers that encode for incorporation of dTTP were prepared by annealing either a 44-mer RNA template or a 44-mer DNA template (of the same sequence) to a 21-mer DNA primer (r44:d21-mer and d44:d21-mer, respectively). The values of the above kinetic constants were determined for dTMP and 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP) incorporation into both template primers. The kcat and Km values calculated from these kinetic constants were similar to the values directly determined from steady-state experiments. Further, the net rate constants for processive incorporation of three successive nucleotides into the r44:d21-mer were similar indicating that a rate-determining step did not follow catalysis. A 20-fold difference in the rate constants (kp) for incorporation of dTMP into the r44:d21-mer versus the d44:d21-mer was largely responsible for the difference in the calculated processivity numbers of 340 and 5, respectively. Finally, the rate constant for pyrophosphorolysis of the 3'-AZTMP-terminated r44:d21-mer (kpyro) was similar to the rate constant for dissociation of the chain-terminated template primer from the enzyme (koff) indicating that millimolar concentrations of intracellular inorganic pyrophosphate would be required for pyrophosphorolysis of AZTMP-terminated retroviral genomes. |
| Author | Reardon, J E |
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| SubjectTerms | Base Sequence Diphosphates - metabolism DNA, Viral - biosynthesis DNA, Viral - chemistry HIV Reverse Transcriptase HIV-1 - enzymology Kinetics Molecular Sequence Data Nucleotides - metabolism Polymers RNA, Viral - metabolism RNA-Directed DNA Polymerase - metabolism |
| Title | Human immunodeficiency virus reverse transcriptase. A kinetic analysis of RNA-dependent and DNA-dependent DNA polymerization |
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