Objective

Protein Arginine Methyltransferase 5 (PRMT5) is a well validated epigenetic target, with several drug candidates entering the clinic. PRMT5 catalyses symmetric dimethylation of arginine residues of histone and non-histone proteins and uses S-Adenosyl methionine (SAM) as a methyl donor. Inhibition of PRMT5 activity can be achieved by small molecules with different Mode of Action (MOA), e.g. SAM competitive or uncompetitive. Understanding the kinetic mechanism of a target allows identification of the different enzyme forms present that could be susceptible for small-molecule inhibition. Pre-steady state kinetics revealed a burst phase of methylation activity indicating a slow product release step. Substrate matrix, as well as dead-end and product inhibition matrix experiments, conclude that PRMT5 uses a Rapid Equilibrium Random mechanism with dead-end EAP and EBQ complexes.