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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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Kinetics of AZT-TP incorporation by Pol γ. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and then rapidly mixed with Mg2+ and various concentrations of AZT-TP [25 (open circle), 75 (filled circle), 150 (open square), 250 (filled square), 375 (open triangle) and 500 μM (filled triangle)]. Each data set was fitted using a single exponential equation to obtain the observed rate and reaction amplitude. (B) The reaction amplitudes were plotted as a function of AZT-TP concentration (filled circle). The data were fitted by non-linear regression using Equation (2) to yield an apparent Kd of 160 ± 40 μM and maximum amplitude of 59 ± 7 nM. The observed rates were also plotted as a function of AZT-TP concentration (open square) and analyzed according to Equation (3) to yield an apparent Kd of 25 ± 10 μM, an overall predicted decrease in the observed rate of 0.95 ± 0.23 s−1, and a Y-intercept of 1.1 ± 0.2 s−1.
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Figure 1: Kinetics of AZT-TP incorporation by Pol γ. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and then rapidly mixed with Mg2+ and various concentrations of AZT-TP [25 (open circle), 75 (filled circle), 150 (open square), 250 (filled square), 375 (open triangle) and 500 μM (filled triangle)]. Each data set was fitted using a single exponential equation to obtain the observed rate and reaction amplitude. (B) The reaction amplitudes were plotted as a function of AZT-TP concentration (filled circle). The data were fitted by non-linear regression using Equation (2) to yield an apparent Kd of 160 ± 40 μM and maximum amplitude of 59 ± 7 nM. The observed rates were also plotted as a function of AZT-TP concentration (open square) and analyzed according to Equation (3) to yield an apparent Kd of 25 ± 10 μM, an overall predicted decrease in the observed rate of 0.95 ± 0.23 s−1, and a Y-intercept of 1.1 ± 0.2 s−1.

Mentions: We previously reported that the single nucleotide incorporation of AZT-TP catalyzed by Pol γ under single turnover conditions was complex compared to that of the other NRTIs examined (17). The kinetics indicated that the amount of product formed in a single turnover was a function of the concentration of AZT-TP in solution. This implied that product formation was reversibly linked to substrate binding. Given the importance of AZT in treating HIV infections and the current debate over the site of the observed clinical toxicities, we thought that it was important first to exclude the possibility that the abnormal kinetics were a result of an artifact due to the incomplete or insufficient quenching of enzyme activity upon the addition of 0.5 M EDTA, as was routinely employed. Therefore, we carried out a similar series of reactions, but instead quenched with a final concentration of 0.5 M HCl. The results are shown in Figure 1A. The data appear to be similar to our previous data, in that, the amplitude of product formation increased in a hyperbolic fashion as a function of AZT-TP concentration (Figure 1B). A fit of the data using a hyperbolic function [Equation (2)] defined an apparent Kd for AZT-TP binding of 160 ± 40 μM and a maximum amplitude of 59 ± 7 nM. In addition, we observed a decrease in the observed rate of incorporation over the concentration series, although the rate measurements at the two lower concentrations were less accurate because of the lower amplitudes. The observed rates of incorporation appeared to decrease hyperbolically over the concentration series and were therefore fitted using Equation (3) to obtain an apparent Kd of 25 ± 10 μM and an overall decrease in the observed rate from ∼1.1 to 0.2 s−1 (net change of 0.95 ± 0.23 s−1). This experiment shows that there is no obvious artifact due to using EDTA to quench the reaction and confirms our previous observation that the kinetics are complex and not explained by the conventional model.Figure 1.


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

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

Kinetics of AZT-TP incorporation by Pol γ. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and then rapidly mixed with Mg2+ and various concentrations of AZT-TP [25 (open circle), 75 (filled circle), 150 (open square), 250 (filled square), 375 (open triangle) and 500 μM (filled triangle)]. Each data set was fitted using a single exponential equation to obtain the observed rate and reaction amplitude. (B) The reaction amplitudes were plotted as a function of AZT-TP concentration (filled circle). The data were fitted by non-linear regression using Equation (2) to yield an apparent Kd of 160 ± 40 μM and maximum amplitude of 59 ± 7 nM. The observed rates were also plotted as a function of AZT-TP concentration (open square) and analyzed according to Equation (3) to yield an apparent Kd of 25 ± 10 μM, an overall predicted decrease in the observed rate of 0.95 ± 0.23 s−1, and a Y-intercept of 1.1 ± 0.2 s−1.
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Related In: Results  -  Collection

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Figure 1: Kinetics of AZT-TP incorporation by Pol γ. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and then rapidly mixed with Mg2+ and various concentrations of AZT-TP [25 (open circle), 75 (filled circle), 150 (open square), 250 (filled square), 375 (open triangle) and 500 μM (filled triangle)]. Each data set was fitted using a single exponential equation to obtain the observed rate and reaction amplitude. (B) The reaction amplitudes were plotted as a function of AZT-TP concentration (filled circle). The data were fitted by non-linear regression using Equation (2) to yield an apparent Kd of 160 ± 40 μM and maximum amplitude of 59 ± 7 nM. The observed rates were also plotted as a function of AZT-TP concentration (open square) and analyzed according to Equation (3) to yield an apparent Kd of 25 ± 10 μM, an overall predicted decrease in the observed rate of 0.95 ± 0.23 s−1, and a Y-intercept of 1.1 ± 0.2 s−1.
Mentions: We previously reported that the single nucleotide incorporation of AZT-TP catalyzed by Pol γ under single turnover conditions was complex compared to that of the other NRTIs examined (17). The kinetics indicated that the amount of product formed in a single turnover was a function of the concentration of AZT-TP in solution. This implied that product formation was reversibly linked to substrate binding. Given the importance of AZT in treating HIV infections and the current debate over the site of the observed clinical toxicities, we thought that it was important first to exclude the possibility that the abnormal kinetics were a result of an artifact due to the incomplete or insufficient quenching of enzyme activity upon the addition of 0.5 M EDTA, as was routinely employed. Therefore, we carried out a similar series of reactions, but instead quenched with a final concentration of 0.5 M HCl. The results are shown in Figure 1A. The data appear to be similar to our previous data, in that, the amplitude of product formation increased in a hyperbolic fashion as a function of AZT-TP concentration (Figure 1B). A fit of the data using a hyperbolic function [Equation (2)] defined an apparent Kd for AZT-TP binding of 160 ± 40 μM and a maximum amplitude of 59 ± 7 nM. In addition, we observed a decrease in the observed rate of incorporation over the concentration series, although the rate measurements at the two lower concentrations were less accurate because of the lower amplitudes. The observed rates of incorporation appeared to decrease hyperbolically over the concentration series and were therefore fitted using Equation (3) to obtain an apparent Kd of 25 ± 10 μM and an overall decrease in the observed rate from ∼1.1 to 0.2 s−1 (net change of 0.95 ± 0.23 s−1). This experiment shows that there is no obvious artifact due to using EDTA to quench the reaction and confirms our previous observation that the kinetics are complex and not explained by the conventional model.Figure 1.

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