UBC Theses and Dissertations
Oligomerization dependent enzyme kinetics and mechanistic characterization of type I protein arginine N-methyltransferases Thomas, Dylan
Protein arginine N-methyltransferases (PRMTs) constitute a family of post-translational modifying enzymes that modulate protein-protein interactions via the addition of methyl groups to arginine residues in protein substrates (1). PRMTs have been demonstrated to homooligomerize via a dimerization arm that binds with the outer surface of the S-adenosyl-Lmethionine (AdoMet) binding domain (2-5). In this body of work, I have demonstrated and quantified in vitro the strength of homodimerization for PRMT1 and PRMT6 and demonstrated that saturating concentrations of S-adenosyl-L-methionine (AdoMet) or S-adenosyl-Lhomocysteine (AdoHcy) respectively strengthen or weaken this interaction. This finding supports an ordered bisubstrate mechanism in which AdoMet binding promotes formation of the complete peptide-substrate binding groove through dimerization, and AdoHcy generation promotes dissociation of the dimeric complex and turnover of substrate. A kinetic study using HIV Tat peptides revealed oligomerization-dependent kinetic patterns with these substrates. Kinetic experiments were initially performed on HIV Tat peptide with novel ωN-substitutions to probe their ability to inhibit PRMT1, 4 and 6. It was found that these Tat-peptides act as substrate inhibitors for both PRMT1 and PRMT6 and that this substrate inhibition was mitigated as the enzyme concentration increased. A model was proposed that represents activity as the sum of each ordered oligomer in solution, with the monomer being uniquely susceptible to substrate inhibition. Diverging from strictly oligomerization effects, R1 fibrillarin-like peptide containing a single arginine was substituted to alter the pKa of the terminal guanidino group to better probe the physicochemical properties that control methyltransfer. Surprisingly, hydroxyl substituted R1 peptide demonstrated an enhanced catalytic constant with PRMT1. MS and MS² experiments demonstrate that only monomethylation occurs on substituted arginines with PRMT1, and that this addition is asymmetric. PRMT1 D51N, a catalytically compromised mutant, revealed the kcat as rate limiting in the presence of D₂O, and electrostatic potential maps indicate that deprotonation of hydroxyl substituted arginine produces a strong nucleophile capable of enhanced methyltransfer. Altogether, these studies support water mediated, ordered bisubstrate mechanism in which oligomerization modulates activity. Substrate inhibition and active site chemistry were investigated using novel chemically substituted peptide probes that highlight trends beyond what site-directed mutagenesis can reveal alone.
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