Catalytic Asymmetry in Homodimeric H+-Pumping Membrane Pyrophosphatase Demonstrated by Non-Hydrolyzable Pyrophosphate Analogs
Abstract
:1. Introduction
2. Results
2.1. Kinetics of Dh-mPPase Inhibition by Substrate Analogs Demonstrates Nonequivalent Active Sites
2.2. Metal Cofactors Modulate Catalytic Asymmetry in mPPase
2.3. Membrane Role in Catalytic Asymmetry
3. Discussion
3.1. Catalytic Asymmetry in mPPase
3.2. The Roles of the Metal Cofactors in the Catalytic Asymmetry
4. Materials and Methods
4.1. Materials
4.2. PPi Hydrolysis Assay
4.3. Calculations and Data Treatment
5. Conclusions and Perspectives
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Parameter | Value | |||
---|---|---|---|---|
IDP | HEDP | AMDP | IDP (Na) | |
A1 (µmol·min−1·mg−1) | 1.32 ± 0.01 | 1.31 ± 0.02 | 1.34 ± 0.02 | 0.27 ± 0.01 |
A2 (µmol·min−1·mg−1) | 0.40 ± 0.04 | 0.37 ± 0.04 | 0.49 ± 0.02 | <0.20 |
A3 (µmol·min−1·mg−1) | 0.53 ± 0.05 | 0.51 ± 0.02 | 0.23 ± 0.03 | 0.07 ± 0.04 |
Ki1 (µM) | 4.2 ± 0.2 | 27 ± 1 | 0.40 ± 0.02 | 2.2 ± 0.2 |
Ki2 (µM) | >500 | >10,000 | 12.5 ± 2.5 | >500 |
Ki(s) (µM) | 90 ± 10 | 470 ± 30 | 4.4 ± 0.4 | 110 ± 30 |
Km1 (µM) | 2.5 ± 0.1 | 2.3 ± 0.1 | 2.6 ± 0.1 | 1.4 ± 0.1 |
Km2 (µM) | 460 ± 60 | 490 ± 50 | 280 ± 30 | >1000 |
Km(i) * (µM) | 53 ± 6 | 40 ± 3 | 27 ± 3 | 70 ± 20 |
RMSD (%) | 3.46 | 3.62 | 3.39 | 7.08 |
Parameter | Value | |
---|---|---|
Scheme 3 | Scheme 3 without M2ES | |
A1 (µmol·min−1·mg−1) | 1.27 ± 0.02 | 1.27 ± 0.02 |
A2 (µmol·min−1·mg−1) | 0.40 ± 0.06 | 0.40 ± 0.05 |
A3 (µmol·min−1·mg−1) | 0.60 ± 0.05 | 0.62 ± 0.05 |
KA1 (µM2) | 0.004 ± 0.002 | - |
KA2 (µM2) | 0.47 ± 0.09 | 0.53 ± 0.09 |
KA3 (µM2) | 0.17 ± 0.12 | - |
KA4 (µM2) | 0.10 ± 0.01 | 0.10 ± 0.01 |
KA1KA3 (µM4) | 0.00057 ± 0.00002 | 0.00060 ± 0.00002 |
KA2KA4 (µM4) | 0.05 ± 0.01 | 0.05 ± 0.01 |
Km1 (µM) | 2.2 ± 0.1 | 2.17 ± 0.05 |
Km2 (µM) | 420 ± 70 | 430 ± 80 |
Km3 * (µM) | 5 ± 1 | |
RMSD (%) | 3.80 | 3.82 |
Ligand | Alkali Metal Ion (50 mM) | Dissociation Constant, mM | |
---|---|---|---|
KML | KM2L | ||
PPi | K+ | 0.112 | 2.84 |
PPi | Na+ | 0.122 | 2.84 |
HEDP | K+ | 0.200 | 1.79 |
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Anashkin, V.A.; Malinen, A.M.; Bogachev, A.V.; Baykov, A.A. Catalytic Asymmetry in Homodimeric H+-Pumping Membrane Pyrophosphatase Demonstrated by Non-Hydrolyzable Pyrophosphate Analogs. Int. J. Mol. Sci. 2021, 22, 9820. https://doi.org/10.3390/ijms22189820
Anashkin VA, Malinen AM, Bogachev AV, Baykov AA. Catalytic Asymmetry in Homodimeric H+-Pumping Membrane Pyrophosphatase Demonstrated by Non-Hydrolyzable Pyrophosphate Analogs. International Journal of Molecular Sciences. 2021; 22(18):9820. https://doi.org/10.3390/ijms22189820
Chicago/Turabian StyleAnashkin, Viktor A., Anssi M. Malinen, Alexander V. Bogachev, and Alexander A. Baykov. 2021. "Catalytic Asymmetry in Homodimeric H+-Pumping Membrane Pyrophosphatase Demonstrated by Non-Hydrolyzable Pyrophosphate Analogs" International Journal of Molecular Sciences 22, no. 18: 9820. https://doi.org/10.3390/ijms22189820
APA StyleAnashkin, V. A., Malinen, A. M., Bogachev, A. V., & Baykov, A. A. (2021). Catalytic Asymmetry in Homodimeric H+-Pumping Membrane Pyrophosphatase Demonstrated by Non-Hydrolyzable Pyrophosphate Analogs. International Journal of Molecular Sciences, 22(18), 9820. https://doi.org/10.3390/ijms22189820