Transient Kinetics Define a Complete Kinetic Model for Protein Arginine Methyltransferase 1.
Protein arginine methyltransferases (PRMTs) are the enzymes responsible for posttranslational methylation of protein arginine residues in eukaryotic cells, particularly within the histone tails. A detailed mechanistic model of PRMT-catalyzed methylation is currently lacking, but it is essential for understanding the functions of PRMTs in various cellular pathways and for efficient design of PRMT inhibitors as potential treatments for a range of human diseases. In this work, we used stopped-flow fluorescence in combination with global kinetic simulation to dissect the transient kinetics of PRMT1, the predominant type I arginine methyltransferase. Several important mechanistic insights were revealed. The cofactor and the peptide substrate bound to PRMT1 in a random manner and then followed a kinetically preferred pathway to generate the catalytic enzyme-cofactor-substrate ternary complex. Product release proceeded in an ordered fashion, with peptide dissociation followed by release of the byproduct S-adenosylhomocysteine. Importantly, the dissociation rate of the monomethylated intermediate from the ternary complex was much faster than the methyl transfer. Such a result provided direct evidence for distributive arginine dimethylation, which means the monomethylated substrate has to be released to solution and rebind with PRMT1 before it undergoes further methylation. In addition, cofactor binding involved a conformational transition, likely an open-to-closed conversion of the active site pocket. Further, the histone H4 peptide bound to the two active sites of the PRMT1 homodimer with differential affinities, suggesting a negative cooperativity mechanism of substrate binding. These findings provide a new mechanistic understanding of how PRMTs interact with their substrates and transfer methyl groups.
From the Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia 30602 and.
Hao
H
Hu
the State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Cheng
C
Luo
From the Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia 30602 and yzheng@uga.edu.
Y George
YG
Zheng
D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011484:Protein-Arginine N-Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D006657:Histones; D050505:Mutant Proteins; D010446:Peptide Fragments; D012097:Repressor Proteins; D001120:Arginine; D012435:S-Adenosylhomocysteine; C099988:PRMT1 protein, human; D011484:Protein-Arginine N-Methyltransferases
PRMT; enzyme catalysis; histone methylation; ordered and random binding mechanism; pre-steady-state kinetics; processivity; protein methylation; stopped-flow; transient kinetics