Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase
Abstract
:1. Introduction
2. Results
2.1. Selection of Residues for Substitution
2.2. Effects of Substitutions on Tetramer Activity and Stability
2.3. Regulation of dhPPase Variants by Adenosine Phosphates
2.4. AMP/ADP and ADP/Ap4A Competition in Variants with Unusual ADP Effects
2.5. Measurements of Adenosine Phosphate Binding by Isothermal Titration Calorimetry (ITC)
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Enzyme Activity Assay
4.3. Isothermal Titration Calorimetry (ITC)
4.4. Structure Modeling and Docking
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Enzyme Variant | AT, % | kd, h−1 | ka, μM−1·h−1 | Kd, μM |
---|---|---|---|---|
WT | 100 ± 5 | 1.6 ± 0.1 (1.8 ± 0.1) a | 2.1 ± 0.5 (2.4 ± 0.4) a | 0.8 ± 0.2 (0.7 ± 0.2) a |
K95A | 66 ± 2 | 1.00 ± 0.04 | 0.3 ± 0.1 | 3.2 ± 0.8 |
K100A | 42 ± 1 | 1.1 ± 0.1 | 0.19 ± 0.04 | 5.9 ± 1.6 |
V117A | 113 ± 1 | 0.44 ± 0.02 | 1.5 ± 0.2 | 0.3 ± 0.1 |
V117T | 141 ± 5 | 0.6 ± 0.1 | 0.6 ± 0.3 | 1.0 ± 0.6 |
G118A | 120 ± 3 | 1.06 ± 0.04 | 0.15 ± 0.04 | 6.8 ± 0.6 |
G118S | 117 ± 2 | 0.85 ± 0.04 | 0.14 ± 0.06 | 6.0 ± 2.7 |
G118M | 21 ± 1 | 0.70 ± 0.04 | 0.18 ± 0.10 | 4 ± 2 |
G118I | 73 ± 1 | 0.75 ± 0.03 | 0.12 ± 0.05 | 6.2 ± 2.9 |
S121A | 120 ± 3 | 1.5 ± 0.1 | 0.5 ± 0.1 | 3.3 ± 0.9 |
Y124A | 83 ± 2 | 1.0 ± 0.1 | 0.9 ± 0.1 | 1.1 ± 0.2 |
E126A | 82 ± 2 | 0.7 ± 0.1 | 3.4 ± 0.5 | 0.21 ± 0.05 |
Enzyme Variant | Adenosine Phosphate and Parameter Value a | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
AMP | ADP | ATP | Ap4A | |||||||||
A1, % | K, µM | h | A1, % | K, µM | h | A1, % | K, µM | h | A1, % | K, nM | h | |
WT b | 3.7 ± 0.1 | 1.4 ± 0.1 | 1.7 ± 0.1 | 9 ± 1 | 0.95 ± 0.05 | 1.34 ± 0.03 | 250 ± 20 | 1.9 ± 0.2 | 1.45 ± 0.13 | 300 ± 10 | 4.9 ± 0.2 | 1.00 ± 0.02 |
K95A | 1.4 ± 0.2 | 2.90 ± 0.04 | 1.7 ± 0.1 | N.A. c | N.A. | N.A. | 317 ± 5 | 3.9 ± 0.1 | 1.63 ± 0.06 | 341 ± 6 | 10.9 ± 0.3 | 1.00 ± 0.02 |
K100A | 2.3 ± 0.4 | 1.82 ± 0.02 | 3.5 ± 0.1 | 750 ± 30 | 7.8 ± 0.2 | 1.55 ± 0.05 | 650 ± 20 | 5.6 ± 0.1 | 1.26 ± 0.04 | 1130 ± 20 | 11.5 ± 0.1 | 1.07 ± 0.01 |
V117A | 66 ± 1 | 124 ± 5 | 1.4 ± 0.1 | 31 ± 1 | 13.4 ± 0.5 | 1.60 ± 0.08 | 138 ± 1 | 0.95 ± 0.05 | 1.22 ± 0.07 | 145 ± 2 | 120 ± 10 | 1.06 ± 0.09 |
V117T | 46 ± 1 | 13.2 ± 0.4 | 1.9 ± 0.1 | 21 ± 1 | 7.9 ± 0.3 | 1.48 ± 0.07 | 165 ± 4 | 0.17 ± 0.02 | 1.34 ± 0.12 | 202 ± 2 | 1.30 ± 0.05 | 1.02 ± 0.04 |
G118A | 0.8 ± 0.7 | 8.4 ± 0.2 | 1.9 ± 0.1 | N.A. | N.A. | N.A. | 156 ± 2 | 2.00 ± 0.07 | 1.92 ± 0.12 | 219 ± 4 | 2.3 ± 0.2 | 1.02 ± 0.06 |
G118S | 0.5 ± 0.6 | 2.55 ± 0.04 | 2.0 ± 0.1 | N.A. | N.A. | N.A. | 277 ± 5 | 0.82 ± 0.03 | 1.25 ± 0.05 | 312 ± 11 | 1.9 ± 0.2 | 1.03 ± 0.08 |
G118M | 3.4 ± 0.6 | 0.76 ± 0.02 | 1.9 ± 0.1 | N.A. | N.A. | N.A. | 314 ± 8 | 4.9 ± 0.2 | 1.70 ± 0.12 | 870 ± 30 | 28 ± 1 | 0.99 ± 0.02 |
G118I | 1.6 ± 1.0 | 2.90 ± 0.03 | 2.2 ± 0.1 | 240 ± 3 | 17 ± 1 | 1.5 ± 0.1 | 410 ± 12 | 6.2 ± 0.2 | 1.30 ± 0.06 | 393 ± 9 | 4.3 ± 0.2 | 1.05 ± 0.04 |
S121A | 1 ± 4 | 1.60 ± 0.02 | 1.9 ± 0.1 | 4.2 ± 0.9 | 2.8 ± 0.1 | 2.0 ± 0.10 | 209 ± 2 | 1.27 ± 0.03 | 1.41 ± 0.05 | 247 ± 4 | 1.6 ± 0.1 | 0.97 ± 0.05 |
Y124A | 2.6 ± 0.6 | 5.4 ± 0.1 | 1.6 ± 0.1 | 3.8 ± 0.6 | 5.0 ± 0.1 | 1.63 ± 0.04 | 299 ± 4 | 2.81 ± 0.06 | 1.40 ± 0.04 | 311 ± 7 | 1.8 ± 0.1 | 1.04 ± 0.05 |
E126A | 0.2 ± 0.9 | 22.5 ± 0.5 | 1.8 ± 0.1 | 6.5 ± 1.6 | 6.2 ± 0.3 | 1.6 ± 0.1 | 172 ± 2 | 0.80 ± 0.06 | 1.25 ± 0.10 | 217 ± 4 | 2.9 ± 0.2 | 1.02 ± 0.07 |
Enzyme Variant | A1, % | A2, % | K1, μM | K2, μM | h1 | h2 |
---|---|---|---|---|---|---|
K95A | 170 ± 30 | 18 ± 2 | 1.7 ± 0.6 | 7 ± 2 | 1.8 ± 0.3 | 1.4 ± 0.2 |
G118A | 180 ± 20 | 91 ± 5 | 4.8 ± 1.5 | 50 ± 20 | 1.5 ± 0.2 | 1.3 ± 0.2 |
G118S | 270 ± 30 | 0.8 ± 1.1 | 0.8 ± 0.2 | 3.0 ± 0.6 | 1.1 ± 0.1 | 1.1 ± 0.1 |
G118M | 174 ± 19 | 10 ± 1 | 1.0 ± 0.5 | 10 ± 2 | 0.9 ± 0.1 | 0.9 ± 0.1 |
Enzyme Variant | Adenosine Phosphate | ΔH, kcal/mol | n | KN, µM | −TΔS, kcal/mol |
---|---|---|---|---|---|
WT | AMP | −5.6 ± 0.5 | 0.79 ± 0.05 | 0.8 ± 0.3 | −2.7 ± 0.9 |
K95A | AMP | −2.6 ± 0.2 | 2.41 ± 0.04 | 4.6 ± 0.7 | −4.7 ± 0.4 |
K100A | AMP | −4.3 ± 0.1 | 1.85 ± 0.04 | 1.3 ± 0.2 | −3.7 ± 0.3 |
V117A | AMP | −4.1 ± 0.6 | 0.94 ± 0.12 | 4.4 ± 0.8 | −3.2 ± 0.8 |
V117T | AMP | −5.5 ± 0.2 | 0.94 ± 0.03 | 1.7 ± 0.2 | −2.4 ± 0.3 |
G118A | AMP | −2.2 ± 0.1 | 1.96 ± 0.09 | 4.2 ± 0.6 | −5.2 ± 0.2 |
G118S | AMP | −3.6 ± 0.2 | 1.16 ± 0.06 | 2.8 ± 0.4 | −4.0 ± 0.3 |
G118M | AMP | −4.0 ± 0.1 | 0.98 ± 0.02 | 0.52 ± 0.07 | −4.5 ± 0.2 |
G118I | AMP | 4.6 ± 0.2 | 1.07 ± 0.03 | 1.6 ± 0.2 | −3.3 ± 0.3 |
S121A | AMP | −5.1 ± 0.2 | 1.07 ± 0.02 | 1.1 ± 0.10 | −3.1 ± 0.3 |
Y124A | AMP | −3.0 ± 0.2 | 1.10 ± 0.04 | 2.5 ± 0.2 | −4.7 ± 0.2 |
E126A | AMP | −3.8 ± 0.2 | 1.05 ± 0.04 | 1.9 ± 0.4 | −4.0 ± 0.4 |
WT | ADP | −5.9 ± 0.3 | 0.90 ± 0.04 | 1.0 ± 0.2 | −2.3 ± 0.5 |
K95A | ADP | −3.6 ± 0.2 | 2.1 ± 0.10 | 9.3 ± 1.7 | −3.2 ± 0.4 |
K100A | ADP | −1.6 ± 0.2 | 2.1 ± 0.2 | 6 ± 2 | −5.5 ± 0.5 |
G118A | ADP | −3.3 ± 0.3 | 2.1 ± 0.1 | 5 ± 1 | −4.0 ± 0.5 |
G118S | ADP | −5.8 ± 0.2 | 1.18 ± 0.03 | 1.1 ± 0.2 | −2.3 ± 0.4 |
E126A | ADP | −5.9 ± 0.2 | 0.97 ± 0.02 | 0.6 ± 0.1 | −2.6 ± 0.3 |
WT | ATP | −6.8 ± 0.2 | 0.88 ± 0.02 | 2.1 ± 0.3 | −0.9 ± 0.3 |
K95A | ATP | −4.3 ± 0.7 | 2.4 ± 0.3 | 6 ± 2 | −2.8 ± 1.0 |
K100A | ATP | −0.41 ± 0.03 | 1.9 ± 0.1 | 0.4 ± 0.2 | −8.3 ± 0.5 |
G118A | ATP | −1.3 ± 0.4 | 2.0 ± 0.3 | 3 ± 4 | −6.3 ± 1.7 |
G118S | ATP | −1.0 ± 0.2 | 1.1 ± 0.1 | 0.5 ± 0.5 | −8 ± 1 |
E126A | ATP | −4.1 ± 0.6 | 1.0 ± 0.1 | 4.3 ± 1.2 | −3.2 ± 0.9 |
WT | Ap4A | −10.3 ± 0.3 | 0.41 ± 0.01 | N.D. b | N.A. c |
K95A | Ap4A | −13.7 ± 0.6 | 0.45 ± 0.01 | N.D. | N.A. |
K100A | Ap4A | −11.4 ± 0.4 | 0.52 ± 0.01 | N.D. | N.A. |
V117A | Ap4A | −9.0 ± 0.3 | 0.41 ± 0.01 | N.D. | N.A. |
V117T | Ap4A | −9.1 ± 0.4 | 0.41 ± 0.01 | N.D. | N.A. |
G118A | Ap4A | −10.0 ± 0.2 | 0.47 ± 0.01 | N.D. | N.A. |
G118S | Ap4A | −8.0 ± 0.2 | 0.40 ± 0.01 | N.D. | N.A. |
G118M | Ap4A | −9.2 ± 0.4 | 0.45 ± 0.01 | N.D. | N.A. |
G118I | Ap4A | −10.0 ± 0.4 | 0.44 ± 0.01 | N.D. | N.A. |
S121A | Ap4A | −14.3 ± 0.3 | 0.42 ± 0.01 | N.D. | N.A. |
Y124A | Ap4A | −10.0 ± 0.4 | 0.41 ± 0.01 | N.D. | N.A. |
E126A | Ap4A | −10.7 ± 0.2 | 0.40 ± 0.01 | N.D. | N.A. |
Residue | Direction of ADP Effect | Effect Size | Binding Stoichiometry | Binding Affinity | Binding Cooperativity | Tetramer Stability |
---|---|---|---|---|---|---|
Lys95 | ++ | +++ | − − | |||
Lys100 | +++ | +++ | +++ | +++ | − − | |
Val117 | + | − − − | ++ | + | ||
Gly118 | +++ | ++ | + | + | +++ | − − |
Ser121 | − | |||||
Tyr124 | + | |||||
Glu126 | + | + |
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Anashkin, V.A.; Kirillova, E.A.; Orlov, V.N.; Baykov, A.A. Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase. Int. J. Mol. Sci. 2024, 25, 5768. https://doi.org/10.3390/ijms25115768
Anashkin VA, Kirillova EA, Orlov VN, Baykov AA. Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase. International Journal of Molecular Sciences. 2024; 25(11):5768. https://doi.org/10.3390/ijms25115768
Chicago/Turabian StyleAnashkin, Viktor A., Elena A. Kirillova, Victor N. Orlov, and Alexander A. Baykov. 2024. "Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase" International Journal of Molecular Sciences 25, no. 11: 5768. https://doi.org/10.3390/ijms25115768