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A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase.

Hanes JW, Johnson KA - Nucleic Acids Res. (2007)

Bottom Line: The kinetics of 3'-azido-2',3'-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked.This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/K(m) (7 x 10(-6) microM(-1) s(-1)).Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5'-triphosphate (8-oxo-dGTP) show this same phenomena.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry & Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas, Austin, TX 78712, USA.

ABSTRACT
Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3'-azido-2',3'-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5'-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s(-1)) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 microM) and chemistry forward and reverse reactions, 0.38 and 0.22 s(-1), respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/K(m) (7 x 10(-6) microM(-1) s(-1)). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5'-triphosphate (8-oxo-dGTP) show this same phenomena.

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Steady-state kinetic parameters of AZT-TP incorporation. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 1.5 μM 25/45-mer DNA (radiolabeled primer) and mixed with Mg2+ and various concentrations of AZT-TP [50 (filled triangle), 100 (open triangle), 200 (filled square), 300 (open square), 400 (filled circle) and 500 (open circle) μM]. The data were fitted by linear regression and the slope divided by the enzyme concentration to obtain the rate of turnover. (B) The rate of turnover was plotted against AZT-TP concentration and the data were analyzed according to the Michaelis–Menton equation to obtain a kcat of 0.001 ± 0.0001 s−1 and a Km of 280 ± 60 μM.
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Figure 5: Steady-state kinetic parameters of AZT-TP incorporation. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 1.5 μM 25/45-mer DNA (radiolabeled primer) and mixed with Mg2+ and various concentrations of AZT-TP [50 (filled triangle), 100 (open triangle), 200 (filled square), 300 (open square), 400 (filled circle) and 500 (open circle) μM]. The data were fitted by linear regression and the slope divided by the enzyme concentration to obtain the rate of turnover. (B) The rate of turnover was plotted against AZT-TP concentration and the data were analyzed according to the Michaelis–Menton equation to obtain a kcat of 0.001 ± 0.0001 s−1 and a Km of 280 ± 60 μM.

Mentions: Under normal circumstances, the specificity constant (kcat/Km) for the incorporation of nucleotides is more accurately obtained using transient state kinetic methods, and because DNA release is slower than incorporation conventional, steady-state methods fail to provide kinetic parameters that accurately reflect nucleotide selectivity during processive synthesis. In the present case, however, our data suggest that pyrophosphate release is much slower than DNA release, and therefore, steady-state methods should provide a valid estimate of kcat/Km. In this unusual case, the relevant kinetic parameters could be determined by measuring the steady-state kinetics of single nucleotide incorporation. The time dependence of product (DNAn+1) formation is shown under steady-state conditions with DNA in excess of Pol γ (Figure 5). The data were analyzed by linear regression, and the observed rate was plotted as a function of concentration defining a kcat of 0.001 ± 0.0001 s−1 and a Km of 280 ± 60 μM. It is important to note that the kcat obtained for this incorporation reaction is 20-fold slower than the rate of DNA release (13). The overall rate of catalysis in this case may be partially limited by a conformational change which limits the release of both PPi and DNAn+1. The specificity constant defined by this experiment is almost 300-fold less ((3.6 ± 0.9) × 10−6 μM−1 s−1) than previously reported (17) or computed from the rate of the single turnover divided by the apparent Kd defined by the amplitude dependence (Figure 1).Figure 5.


A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase.

Hanes JW, Johnson KA - Nucleic Acids Res. (2007)

Steady-state kinetic parameters of AZT-TP incorporation. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 1.5 μM 25/45-mer DNA (radiolabeled primer) and mixed with Mg2+ and various concentrations of AZT-TP [50 (filled triangle), 100 (open triangle), 200 (filled square), 300 (open square), 400 (filled circle) and 500 (open circle) μM]. The data were fitted by linear regression and the slope divided by the enzyme concentration to obtain the rate of turnover. (B) The rate of turnover was plotted against AZT-TP concentration and the data were analyzed according to the Michaelis–Menton equation to obtain a kcat of 0.001 ± 0.0001 s−1 and a Km of 280 ± 60 μM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2175305&req=5

Figure 5: Steady-state kinetic parameters of AZT-TP incorporation. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 1.5 μM 25/45-mer DNA (radiolabeled primer) and mixed with Mg2+ and various concentrations of AZT-TP [50 (filled triangle), 100 (open triangle), 200 (filled square), 300 (open square), 400 (filled circle) and 500 (open circle) μM]. The data were fitted by linear regression and the slope divided by the enzyme concentration to obtain the rate of turnover. (B) The rate of turnover was plotted against AZT-TP concentration and the data were analyzed according to the Michaelis–Menton equation to obtain a kcat of 0.001 ± 0.0001 s−1 and a Km of 280 ± 60 μM.
Mentions: Under normal circumstances, the specificity constant (kcat/Km) for the incorporation of nucleotides is more accurately obtained using transient state kinetic methods, and because DNA release is slower than incorporation conventional, steady-state methods fail to provide kinetic parameters that accurately reflect nucleotide selectivity during processive synthesis. In the present case, however, our data suggest that pyrophosphate release is much slower than DNA release, and therefore, steady-state methods should provide a valid estimate of kcat/Km. In this unusual case, the relevant kinetic parameters could be determined by measuring the steady-state kinetics of single nucleotide incorporation. The time dependence of product (DNAn+1) formation is shown under steady-state conditions with DNA in excess of Pol γ (Figure 5). The data were analyzed by linear regression, and the observed rate was plotted as a function of concentration defining a kcat of 0.001 ± 0.0001 s−1 and a Km of 280 ± 60 μM. It is important to note that the kcat obtained for this incorporation reaction is 20-fold slower than the rate of DNA release (13). The overall rate of catalysis in this case may be partially limited by a conformational change which limits the release of both PPi and DNAn+1. The specificity constant defined by this experiment is almost 300-fold less ((3.6 ± 0.9) × 10−6 μM−1 s−1) than previously reported (17) or computed from the rate of the single turnover divided by the apparent Kd defined by the amplitude dependence (Figure 1).Figure 5.

Bottom Line: The kinetics of 3'-azido-2',3'-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked.This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/K(m) (7 x 10(-6) microM(-1) s(-1)).Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5'-triphosphate (8-oxo-dGTP) show this same phenomena.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry & Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas, Austin, TX 78712, USA.

ABSTRACT
Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3'-azido-2',3'-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5'-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s(-1)) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 microM) and chemistry forward and reverse reactions, 0.38 and 0.22 s(-1), respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/K(m) (7 x 10(-6) microM(-1) s(-1)). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5'-triphosphate (8-oxo-dGTP) show this same phenomena.

Show MeSH
Related in: MedlinePlus