A Kinetic Study of Human Protein Arginine N-Methyltransferase 6 Reveals a Distributive Mechanism

Abstract

Human protein arginine N-methyltransferase 6 (PRMT6) transfers methyl groups from the co-substrate S-adenosyl-Lmethionine to arginine residues within proteins, forming S-adenosyl-L-homocysteine as well as -N G -monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA) residues in the process. We have characterized the kinetic mechanism of recombinant His-tagged PRMT6 using a mass spectrometry method for monitoring the methylation of a series of peptides bearing a single arginine, MMA, or aDMA residue. We find that PRMT6 follows an ordered sequential mechanism in which S-adenosyl-L-methionine binds to the enzyme first and the methylated product is the first to dissociate. Furthermore, we find that the enzyme displays a preference for the monomethylated peptide substrate, exhibiting both lower K m and higher V max values than what are observed for the unmethylated peptide. This difference in substrate K m and V max , as well as the lack of detectable aDMA-containing product from the unmethylated substrate, suggest a distributive rather than processive mechanism for multiple methylations of a single arginine residue. In addition, we speculate that the increased catalytic efficiency of PRMT6 for methylated substrates combined with lower K m values for native protein methyl acceptors may obscure this distributive mechanism to produce an apparently processive mechanism.Human protein arginine N-methyltransferases (PRMTs) 2 are a family of enzymes that transfer methyl groups from the co-substrate S-adenosyl-L-methionine (AdoMet) to the terminal nitrogen atoms on the guanidino groups of arginine residues within proteins, forming S-adenosyl-L-homocysteine

Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger

“…The biological significance of this remains unclear, but MTases are responsible for the methylation of DNA, RNA and proteins and also numerous small molecule metabolites and natural products. PRMT6-catalysed methylation is proposed to affect gene regulation by modifying protein-nucleic acid interactions [39] while the methylation of a 5-methyluridine (m5U) residue at position 54 by tRNA (m5U54)-MTases, is a conserved feature in eukaryotic tRNA which, in vitro, influences the fidelity and rate of protein synthesis as well as the stability of tRNA tertiary structure [40]. The altered expression of these MTases possibly highlight the affect GT has on transcription and translation, as noted by O'Keeffe et al for A. fumigatus ΔgliT [41].…”

TbPRMT6 Is a Type I Protein Arginine Methyltransferase That Contributes to Cytokinesis in Trypanosoma brucei

“…The human enzyme is reported to display an exclusively nuclear localization pattern (28), consistent with its known roles in nuclear processes. Detailed in vitro studies showed that human PRMT6 catalyzes methyl transfers in a distributive manner, depositing the first methyl group and creating MMA, dissociating from the substrate, and then rebinding to the methyl mark and forming ADMA (53). Homologues of PRMT6 are apparently absent from the genomes of most single-celled eukaryotes, with the exception of Trypanosoma brucei and, possibly, Dictyostelium (3).…”

“…The homologues of PRMT6 in humans and other higher eukaryotes comprise a family of type I PRMTs involved in transcription and DNA repair (28,53). PRMT6 exhibits a relatively narrow substrate specificity, with the currently known substrates being HMG1A (66,87,106), histone subunits (32,37,38), DNA polymerase beta (20), and several components of the HIV virus (10,39,40) as well as PRMT6 itself (28).…”

Methylation of the nuclear poly(A)-binding protein by type I protein arginine methyltransferases – how and why

“…However, the discrepancy in dissociation rate constants is puzzling. Wooderchak et al (2008) also found a distributive behavior of PRMT1 with a peptide containing a single arginine residue, and a distributive mechanism has been described for PRTM6 as well (Lakowski and Frankel , 2008 ). In contrast, Osborne et al (2007) reported PRMT1 to be partially processive in the methylation of a single arginine residue, i.e., monomethyl arginine and dimethyl arginine were found in comparable quantities.…”