Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism

Abstract

BACKGROUNDProtein arginine N‐methyltransferases (PRMTs) are responsible for the transfer of one or two methyl groups from the methyl donor S‐adenosyl‐L‐methionine (SAM) to produce mono‐ or dimethylated arginine residues and the spent cofactor S‐adenosyl‐L‐homocysteine (SAH). Methylated arginines in eukaryotic cells are involved in several crucial biological processes, including transcription control, DNA damager repair, and RNA processing. However, contrasting findings for two major questions in the field have generated debate regarding PRMT mechanisms. The purpose of this research was to elucidate the PRMT1 mechanism and answer these two highly debated questions: do PRMTs dimethylate their substrates processively or distributively, and do PRMTs bind their substrates using a random or sequential method of substrate binding?METHODSTo explore these mechanisms, we assayed PRMT1 with well‐characterized peptide substrates. We used tandem mass spectrometry to quantitate the ratios of mono‐ and dimethylarginine species produced to assess PRMT1 processivity. We determined the mechanism of bisubstrate binding using radioactive SAM in enzyme assays to measure methyl transfer to a target peptide in the presence of various concentrations of product inhibitors.RESULTSWe demonstrate that PRMT1 processivity differs depending on the substrate sequence, which is congruent with previous reports. However, we show for the first time that as cofactor and/or enzyme concentration increases, the ratio of dimethylarginine to monomethylarginine substantially increases, suggesting the enzyme becomes more processive. Further, the steady‐state inhibition patterns favour a sequential ordered mechanism over a random mechanism, indicating that for catalysis to occur, SAM binding must precede peptide binding.CONCLUSIONSThe degree of PRMT1 processivity is a multifactorial effect that is heavily influenced by experimental design considerations, so previous conclusions about processivity may require reassessment. We also find that PRMT1 uses a sequential ordered substrate binding mechanism, in contrast with recent findings. Use of nonlinear regression and presentation of our data using three linear plots strengthens our conclusions. Considering the conserved active sites within the human PRMT family, we anticipate that our results regarding PRMT1 kinetic and processive mechanisms may extend to other human PRMTs.Support or Funding InformationThis work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN‐2015–04450 Discovery Grant (A.F.), NSERC CGS‐M (J.I.B.), the Shaughnessy Hospital Volunteer Society Fellowship in Health Care (J.I.B.), and the Walter C. Koerner Fellowship (J.I.B.). Additional support provided by Utrecht University and the Netherlands Organization for Scientific Research is acknowledged (T.K., J.v.S., and N.I.M.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.