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J. Biol. Chem., Vol. 283, Issue 15, 10015-10025, April 11, 2008

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

From the Division of Biomolecular & Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Human protein arginine N-methyltransferase 6 (PRMT6) transfers methyl groups from the co-substrate S-adenosyl-L-methionine 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.

Received for publication, December 13, 2007 , and in revised form, January 15, 2008.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Holds a grant from Canadian Institutes of Health Research (Grant MOP 79271) and is a Canada Research Chair recipient. To whom correspondence should be addressed: Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-7146; Fax: 604-822-3035; E-mail: afrankel{at}interchange.ubc.ca.

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