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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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Determination of the Kd for AZT-TP in competition with TTP. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and then rapidly mixed with Mg2+, 1 μM TTP and various concentrations of AZT-TP [0 (open circle), 50 (filled circle), 200 (open square) and 400 (filled square) μM]. Each data set was fitted using a single exponential equation to obtain the observed rate of incorporation. (B) The observed rates were plotted as a function of AZT-TP concentration and fitted using Equation (3) to obtain an apparent Kd of 53 ± 13 μM for AZT-TP binding, a Y-intercept of 9.7 ± 0.3 s−1, and an overall decrease in the observed rate of 8.6 ± 0.6 s−1. According to Equation (4), a true Kd of 20 ± 7 μM for AZT-TP in competition with TTP can be defined.
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Figure 4: Determination of the Kd for AZT-TP in competition with TTP. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and then rapidly mixed with Mg2+, 1 μM TTP and various concentrations of AZT-TP [0 (open circle), 50 (filled circle), 200 (open square) and 400 (filled square) μM]. Each data set was fitted using a single exponential equation to obtain the observed rate of incorporation. (B) The observed rates were plotted as a function of AZT-TP concentration and fitted using Equation (3) to obtain an apparent Kd of 53 ± 13 μM for AZT-TP binding, a Y-intercept of 9.7 ± 0.3 s−1, and an overall decrease in the observed rate of 8.6 ± 0.6 s−1. According to Equation (4), a true Kd of 20 ± 7 μM for AZT-TP in competition with TTP can be defined.

Mentions: Because the concentration dependence of the kinetics of incorporation of AZT are complex (Figure 1), estimates for the Kd for ground state nucleotide binding of AZT-TP were dependent upon the model used to fit the data. Therefore, we measured the Kd for AZT-TP by competition against TTP in a single nucleotide incorporation. This assay was possible because of the dramatic difference in the rate of incorporation of TTP (kpol ∼ 25 s−1) compared to that for AZT-TP so that AZT-TP binds but is not incorporated on the time scale of a single turnover of incorporation of TTP. The observed rate of TTP incorporation at a concentration of 1 μM (close to the Kd for TTP) was measured in the presence of various concentrations of AZT-TP under single turnover conditions (Figure 4). The observed rate of incorporation decreased hyperbolically as a function of increasing AZT-TP concentration with an apparent Kd of 53 ± 13 μM [Equation (3)]. The Kd for TTP (0.63 μM) was previously measured under these conditions and was used according to the relationship shown in Equation (4) to determine a true Kd of 20 ± 7 μM for AZT-TP. This estimate of the Kd obtained by this method is 8-fold tighter than that predicted previously by analyzing the obvious amplitude change of the single nucleotide incorporation reactions (Figure 1B). However, this estimate appears to mirror the apparent Kd obtained from the hyperbolic fit of the decrease in the observed rate (Figure 1B).Figure 4.


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

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

Determination of the Kd for AZT-TP in competition with TTP. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and then rapidly mixed with Mg2+, 1 μM TTP and various concentrations of AZT-TP [0 (open circle), 50 (filled circle), 200 (open square) and 400 (filled square) μM]. Each data set was fitted using a single exponential equation to obtain the observed rate of incorporation. (B) The observed rates were plotted as a function of AZT-TP concentration and fitted using Equation (3) to obtain an apparent Kd of 53 ± 13 μM for AZT-TP binding, a Y-intercept of 9.7 ± 0.3 s−1, and an overall decrease in the observed rate of 8.6 ± 0.6 s−1. According to Equation (4), a true Kd of 20 ± 7 μM for AZT-TP in competition with TTP can be defined.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Determination of the Kd for AZT-TP in competition with TTP. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and then rapidly mixed with Mg2+, 1 μM TTP and various concentrations of AZT-TP [0 (open circle), 50 (filled circle), 200 (open square) and 400 (filled square) μM]. Each data set was fitted using a single exponential equation to obtain the observed rate of incorporation. (B) The observed rates were plotted as a function of AZT-TP concentration and fitted using Equation (3) to obtain an apparent Kd of 53 ± 13 μM for AZT-TP binding, a Y-intercept of 9.7 ± 0.3 s−1, and an overall decrease in the observed rate of 8.6 ± 0.6 s−1. According to Equation (4), a true Kd of 20 ± 7 μM for AZT-TP in competition with TTP can be defined.
Mentions: Because the concentration dependence of the kinetics of incorporation of AZT are complex (Figure 1), estimates for the Kd for ground state nucleotide binding of AZT-TP were dependent upon the model used to fit the data. Therefore, we measured the Kd for AZT-TP by competition against TTP in a single nucleotide incorporation. This assay was possible because of the dramatic difference in the rate of incorporation of TTP (kpol ∼ 25 s−1) compared to that for AZT-TP so that AZT-TP binds but is not incorporated on the time scale of a single turnover of incorporation of TTP. The observed rate of TTP incorporation at a concentration of 1 μM (close to the Kd for TTP) was measured in the presence of various concentrations of AZT-TP under single turnover conditions (Figure 4). The observed rate of incorporation decreased hyperbolically as a function of increasing AZT-TP concentration with an apparent Kd of 53 ± 13 μM [Equation (3)]. The Kd for TTP (0.63 μM) was previously measured under these conditions and was used according to the relationship shown in Equation (4) to determine a true Kd of 20 ± 7 μM for AZT-TP. This estimate of the Kd obtained by this method is 8-fold tighter than that predicted previously by analyzing the obvious amplitude change of the single nucleotide incorporation reactions (Figure 1B). However, this estimate appears to mirror the apparent Kd obtained from the hyperbolic fit of the decrease in the observed rate (Figure 1B).Figure 4.

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