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

Ted M Lakowski,Adam Frankel

doi:10.1074/jbc.m710176200

Abstract

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 omega-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.

Highlights

As opposed to vides for the kinetic description of a single methylation event, the mixed inhibition observed in Fig. 5C, Fig. 7C shows a pos- whereas the R1(aDMA) peptide serves as its corresponding sible uncompetitive type inhibition of H6PRMT6 at low con- product inhibitor. centrations of R1(aDMA) for which R1 is the Reaction Rate Measurements—Many methods have been variable substrate and the AdoMet concentration is constant. employed for measuring the amount and rate of methylation by at higher concentrations of R1(aDMA) no discern- various methyltransferase enzymes
For the specific case of PRMTs, radioactively methylated pro- We have shown that acid-hydrolyzed R1(MMA) in the prestein products have been hydrolyzed, and the component-meth- ence of [14C]AdoMet produces trace amounts of radioactive ylated arginine amino acids are separated by ion exchange compounds that have RF values consistent with MMA, aDMA, chromatography [42], TLC [8], or reversed-phase high-perfor- sDMA, and some additional unidentified compounds that may mance liquid chromatography [5]
In accordance with the consensus of these studies we find that the simplest mechanism that is consistent with the variable and fixed substrate data (Fig. 4) and the product inhibition data (Fig. 5) is a steady-state ordered sequential mechanism involving an enzyme bi-substrate complex in which AdoMet binds first and the methylated arginine product peptide is the first to dissociate (Fig. 6)

Summary

EXPERIMENTAL PROCEDURES

PRMT6 Enzyme—The construct for His-tagged PRMT6 (H6PRMT6) was generated by subcloning the gene from the pGEX-6P-1 vector (a gift from Dr Steven Clarke at UCLA) into the pET28aϩ vector using the restriction enzymes BamHI and XhoI. To establish a linear range of enzyme activity, 50 ␮M R1 or R1(MMA) substrates were incubated with 200 ␮M. Methylation reactions to establish a linear range for enzyme activity were repeated as above with 50 ␮M R1 or R1(MMA) substrate except that the final reaction volume was 20 ␮l, and 100 ␮M [14C]AdoMet (4.1 kBq per 20-␮l reaction) was used. The reactions were stopped at times 0 –300 min by the addition of 10 ␮l of a 70% suspension of nickel-nitrilotriacetic acid nickel affinity agarose (Qiagen) with 2 ␮l of 2.5 mM AdoHcy, and incubated for 10 min at ambient temperature This reaction was centrifuged and the resulting 20-␮l supernatant was added to Pyrex hydrolysis tubes, followed by the addition of 100 ␮l of acetone. Because the traditional reciprocal plots provide a convenient qualitative assessment of enzyme mechanism for both the initial plots of fixed and varied substrates and for the product inhibition studies, we used this method of analysis throughout

RESULTS

DISCUSSION

Summary of product inhibition studies