Characterization of the Product Specificity and Kinetic Mechanism of Protein Arginine Methyltransferase 1

Protein arginine methyltransferases (PRMTs) aid in the regulation of many biological processes by methylating specific arginyl groups within targeted proteins. The varied nature of the response to methylation is, in part, due to the diverse product specificity displayed by the PRMTs. In addition to site location within a protein, biological response is also determined by the degree (mono/dimethylation) and type of arginine dimethylation (asymmetric/symmetric). Here we have identified two strictly conserved methionine residues in the PRMT1 active site that are not only important for activity, but also control substrate specificity. Mutation of Met155 or Met48 results in a loss in activity and a change in distribution of monoand dimethylated products. The altered substrate specificity of M155A and M48L mutants is also evidenced by automethylation. Investigation into the mechanistic basis of altered substrate recognition led us to consider each methyl transfer step separately. Single turnover experiments reveal that the rate of transfer of the second methyl group is arginine residues located in the center of the peptide much slower than transfer of the first methyl group in Met-48L, especially for substrate where turnover of the monomethylated species 1 S. Gui, W.L. Wooderchak, M.P. Daly, P.J. Porter, S.J. Johnson, and J.M. Hevel, Investigation of the molecular origins of protein-arginine methyltransferase I (PRMT1) product specificity reveals a role for two conserved methionine residues, J. Biol. Chem 286 (2011) 29118–29126 51 is negligible. Thus, altered product specificity in M48L originates from the differential effect of the mutation on the two rates. Characterization of the two active-site methionines provides the first insight into how the PRMT1 active site is engineered to control product specificity.