Mutational and Kinetic Analysis of Lesion Recognition by Escherichia coli Endonuclease VIII
Abstract
:1. Introduction
2. Materials and Methods
2.1. Oligodeoxyribonucleotides (ODNs) and Enzymes
2.2. Stopped-Flow Fluorescence Measurements
2.3. Data Analysis
2.4. An Assay of Relative Enzymatic Activity
3. Results
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Shorthand | Sequence |
---|---|
DHU-substrate | TCTCTC(DHU)CCTTCC AGAGAG G GGAAGG |
AP-substrate | TCTCTC(AP)CCTTCC AGAGAG G GGAAGG |
|
Constants | L70S | L70W | Y71W | F121W | F230W | P253W | WT [7] |
---|---|---|---|---|---|---|---|
k1, M−1s−1 | (0.09 ± 0.02) × 106 | (0.06 ± 0.01) × 106 | (21 ± 3) × 106 | (39 ± 11) × 106 | (30 ± 2) × 106 | (27 ± 2) × 106 | (36 ± 7) × 106 |
k−1, s−1 | 1 ± 0.1 | 0.4 ± 0.1 | 330 ± 30 | 120 ± 31 | 310 ± 45 | 310 ± 26 | 410 ± 20 |
K1, M−1 | 0.9 × 105 | 1.5 × 105 | 0.63 × 105 | 3 × 105 | 0.97 × 105 | 0.87 × 105 | 0.88 × 105 |
k2, s−1 | 16 ± 3 | 17 ± 4 | 21 ± 3 | 23 ± 3 | 27 ± 2 | ||
k−2, s−1 | 2.5 ± 0.1 | 0.55 ± 0.25 | 1.2 ± 0.1 | 0.8 ± 0.1 | 1.8 ± 0.3 | ||
K2 | 6.4 | 31 | 17.5 | 28.7 | 15 | ||
k3, s−1 | 0.4 ± 0.1 | 0.58 ± 0.15 | 0.82 ± 0.04 | 1.0 ± 0.03 | 1.6 ± 0.1 | ||
k−3, s−1 | 1.1 ± 0.1 | 0.89 ± 0.14 | 0.66 ± 0.06 | 1.2 ± 0.1 | 1.5 ± 0.2 | ||
K3 | 0.36 | 0.65 | 1.2 | 0.83 | 1.1 | ||
Kass, M−1 | 1.45 × 105 | 6 × 106 | 2 × 106 | 2 × 106 | 1.45 × 106 | ||
kcat, s−1 | 0.09 ± 0.01 | 0.08 ± 0.03 | 0.14 ± 0.03 | 0.26 ± 0.09 | 0.38 ± 0.01 | 0.4 ± 0.03 | 0.35 ± 0.02 |
Kp, M | (1.0 ± 0.2) × 10−6 | (0.7 ± 0.5) × 10−6 | (0.4 ± 0.1) × 10−6 | (0.6 ± 0.1) × 10−6 | (0.6 ± 0.2) × 10−6 | (0.63 ± 0.08) × 10−6 | (0.7 ± 0.1) × 10−6 |
Constants | L70S | L70W | Y71W | F121W | F230W | P253W | WT [7] |
---|---|---|---|---|---|---|---|
k1, M−1s−1 | (0.09 ± 0.01) × 106 | (0.11 ± 0.02) × 106 | (14 ± 2) × 106 | (16 ± 1) × 106 | (18 ± 8) × 106 | (18 ± 3) × 106 | (12 ± 1) × 106 |
k−1, s−1 | 0.9 ± 0.03 | 0.6 ± 0.05 | 170 ± 17 | 132 ± 12 | 81 ± 32 | 130 ± 28 | 260 ± 10 |
K1, M−1 | 1 × 105 | 1.8 × 105 | 0.8 × 105 | 1.2 × 105 | 2.2 × 105 | 1.4 × 105 | 0.5 × 105 |
k2, s−1 | 21 ± 2 | 35 ± 3 | 25 ± 6 | 37 ± 4 | 30 ± 1 | ||
k−2, s−1 | 1.4 ± 0.1 | 0.9 ± 0.3 | 1.0 ± 0.3 | 1.9 ± 0.5 | 4.4 ± 0.2 | ||
K2 | 15 | 39 | 25 | 19 | 6.8 | ||
k3, s−1 | 0.13 ± 0.01 | 0.8 ± 0.1 | 0.9 ± 0.1 | 0.35 ± 0.02 | 2.1 ± 0.1 | ||
k−3, s−1 | 1.0 ± 0.1 | 3.0 ± 0.6 | 4.7 ± 1.1 | 1.9 ± 0.2 | 1.1 ± 0.1 | ||
K3 | 0.13 | 0.27 | 0.19 | 0.18 | 1.9 | ||
K1K2K3, M−1 | 1 × 105 | 1 × 105 | 1.6 × 105 | 12.6 × 105 | 10.5 × 105 | 4.9 × 105 | 6.5 × 105 |
kcat, s−1 | 0.09 ± 0.01 | 0.08 ± 0.007 | 0.29 ± 0.03 | 0.41 ± 0.05 | 1.0 ± 0.1 | 0.45 ± 0.08 | 0.79 ± 0.03 |
Kp, M | (0.18 ± 0.02) × 10−6 | (0.2 ± 0.1) × 10−6 | (0.11 ± 0.01) × 10−6 | (1.5 ± 0.4) × 10−6 | (1.1 ± 0.6) × 10−6 | (1.4 ± 0.2) × 10−6 | (1.8 ± 0.3) × 10−6 |
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Kladova, O.A.; Kuznetsova, A.A.; Fedorova, O.S.; Kuznetsov, N.A. Mutational and Kinetic Analysis of Lesion Recognition by Escherichia coli Endonuclease VIII. Genes 2017, 8, 140. https://doi.org/10.3390/genes8050140
Kladova OA, Kuznetsova AA, Fedorova OS, Kuznetsov NA. Mutational and Kinetic Analysis of Lesion Recognition by Escherichia coli Endonuclease VIII. Genes. 2017; 8(5):140. https://doi.org/10.3390/genes8050140
Chicago/Turabian StyleKladova, Olga A., Alexandra A. Kuznetsova, Olga S. Fedorova, and Nikita A. Kuznetsov. 2017. "Mutational and Kinetic Analysis of Lesion Recognition by Escherichia coli Endonuclease VIII" Genes 8, no. 5: 140. https://doi.org/10.3390/genes8050140
APA StyleKladova, O. A., Kuznetsova, A. A., Fedorova, O. S., & Kuznetsov, N. A. (2017). Mutational and Kinetic Analysis of Lesion Recognition by Escherichia coli Endonuclease VIII. Genes, 8(5), 140. https://doi.org/10.3390/genes8050140