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

Protein arginine methylation is an essential post-translational modification catalyzed by protein arginine methyltransferases (PRMTs). Type I PRMTs transfer the methyl group from S-adenosyl-L-methionine (AdoMet) to the arginine residues and catalyze the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA). Type II PRMTs generate MMA and symmetric dimethylarginine (SDMA). PRMT-catalyzed methylation is involved in many biological processes and human diseases when dysregulated. As the predominant PRMT, PRMT1 catalyzes an estimated 85% of all protein arginine methylation in vivo. Nevertheless, the product specificity of PRMT1 remains poorly understood. A few articles have been published regarding the kinetic mechanism of PRMT1, yet with controversial conclusions. To gain more insights into the product specificity of PRMT1, we dissected the active site of PRMT1 and identified two conserved methionines (Met-48 and Met-155) significant for the enzymatic activity and the product specificity. These two methionines regulate the final product distribution between MMA and ADMA by differentially affecting the first and second methyl transfer step. Current data show that Met-48 also specifies ADMA formation from SDMA. To further understand the kinetic mechanism of PRMT1, we developed a double turnover experiments to conveniently assay the processivity of the two-step methyl transfer. Using the double turnover experiments, we observed that PRMT1-catalyzed dimethylation is semi-processive. The degree of processivity depends on the substrate sequences, which satisfies the controversy between the distributive or partially processive mechanisms previously reported. We are using transient kinetics and single turnover experiments to further investigate the mechanism of PRMT1. Interestingly, during these studies, we found that PRMT1 may incur oxidative damage and the histidine affinity tag influences the protein characteristics of PRMT1. These studies have given important insights into the product specificity and kinetic mechanism of PRMT1, and provided a strong foundation for future studies on PRMT1