Protein arginine methylation is an important modification of proteins, involved in many cellular processes. Some examples are transcription, RNA editing, cellular communication, DNA repair, viral replication and chromatin remodeling. In recent years, the significance of protein arginine methyltransferases (PRMTs) in human diseases has been increasingly studied, especially in cardiovascular disease and cancer. Although the importance of these enzymes is recognized, the understanding of how exactly PRMTs function is still limited. Very little information is available to explain how or why any of the different PRMTs interact with other proteins or, what determines where in that protein to place their methyl marks. Adding to this complexity, placing one of the three different methylation marks (products) or the other (mono methyl arginine MMA, asymmetric dimethyl ADMA, or symmetric dimethyl SDMA) on a protein can cause a cell to respond differently. Therefore, if we really want to understand how this family of proteins functions and how to control them, it’s essential that we understand how they achieve their product specificity; this means, how they decide which methyl mark to place on an interacting protein. In order to better understand the product specificity of this family of enzymes, I have been using as a model two Protein arginine methyltransferases that are responsible different methylation marks: PRMT1, which can make both ADMA and MMA and TbPRMT7, which can only make MMA. Using the information that crystal structure of these enzymes provide and what we already know about how PRMT activity is regulated, my aim is to better understand the mechanisms by which these enzymes achieve their product specificity.

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.