Characterization of the Kinetic Mechanism of Human Protein Arginine Methyltransferase 5.
Biochemistry
; 59(50): 4775-4786, 2020 12 22.
Article
em En
| MEDLINE
| ID: mdl-33274632
ABSTRACT
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 PRMT5MEP50 complex (hPRMT5MEP50) in insect cells for a detailed mechanistic study. In this report, we carry out steady state, product, and dead-end inhibitor studies that show hPRMT5MEP50 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Limite:
Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article