Characterization of the Kinetic Mechanism of Human Protein Arginine Methyltransferase 5.
Alice R EddershawChristopher J StubbsLucy V EdwardesElizabeth UnderwoodGregory R HammPaul R J DaveyPaul N ClarksonKarl SysonPublished in: Biochemistry (2020)
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.
Keyphrases
- mass spectrometry
- induced apoptosis
- endothelial cells
- amino acid
- cell cycle arrest
- protein protein
- liquid chromatography
- nitric oxide
- gas chromatography
- small molecule
- induced pluripotent stem cells
- binding protein
- high resolution
- endoplasmic reticulum stress
- cell proliferation
- bone mineral density
- body composition
- molecular dynamics
- cell death
- high frequency
- molecular dynamics simulations
- risk assessment
- postmenopausal women
- tandem mass spectrometry
- zika virus
- sensitive detection