Characterization of the Kinetic Mechanism of Human Protein Arginine Methyltransferase 5
| العنوان: | Characterization of the Kinetic Mechanism of Human Protein Arginine Methyltransferase 5 |
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| المؤلفون: | Alice R Eddershaw, Karl Syson, Paul R. J. Davey, Gregory Hamm, Christopher D. Stubbs, Paul Clarkson, Elizabeth Underwood, Lucy V Edwardes |
| المصدر: | Biochemistry. 59:4775-4786 |
| بيانات النشر: | American Chemical Society (ACS), 2020. |
| سنة النشر: | 2020 |
| مصطلحات موضوعية: | Models, Molecular, Protein-Arginine N-Methyltransferases, Methyltransferase, Arginine, In Vitro Techniques, Biochemistry, Mass Spectrometry, Substrate Specificity, 03 medical and health sciences, Protein structure, Catalytic Domain, Humans, Protein Interaction Domains and Motifs, Amino Acid Sequence, Enzyme Inhibitors, Protein Structure, Quaternary, Ternary complex, Adaptor Proteins, Signal Transducing, 0303 health sciences, Chemistry, Protein arginine methyltransferase 5, 030302 biochemistry & molecular biology, Deuterium Exchange Measurement, Recombinant Proteins, Kinetics, Biophysics, Hydrogen–deuterium exchange, Steady state (chemistry) |
| الوصف: | Protein arginine methyltransferases (PRMTs) are of great interest for the development of therapeutics due to their involvement in a number of malignancies, such as lung and colon cancer. PRMT5 catalyzes the formation of symmetrical dimethylarginine of a wide variety of substrates and is responsible for the majority of this mark within cells. To gain insight into the mechanism of PRMT5 inhibition, we co-expressed the human PRMT5:MEP50 complex (hPRMT5:MEP50) in insect cells for a detailed mechanistic study. In this report, we carry out steady state, product, and dead-end inhibitor studies that show hPRMT5:MEP50 uses a rapid equilibrium random order mechanism with EAP and EBQ dead-end complexes. We also provide evidence of ternary complex formation in solution using hydrogen/deuterium exchange mass spectrometry. Isotope exchange and intact protein mass spectrometry further rule out ping-pong as a potential enzyme mechanism, and finally, we show that PRMT5 exhibits a pre-steady state burst that corresponds to an initial slow turnover with all four active sites of the hetero-octamer being catalytically active. |
| تدمد: | 1520-4995 0006-2960 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e0c63875119c271f482688a035fe6bfcTest https://doi.org/10.1021/acs.biochem.0c00554Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....e0c63875119c271f482688a035fe6bfc |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15204995 00062960 |
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