A fast and efficient method for quantitative measurement of S-adenosyl-l-methionine-dependent methyltransferase activity with protein substrates

Background:The protein-lysine methyltransferase LSMT from pea methylates the large subunit of Rubisco. Results: In Arabidopsis, Rubisco is not methylated, and the physiological substrates of the LSMT-like enzyme are chloroplastic aldolases. Conclusion: LSMT homologs from plants display different substrate specificities, with targets involved in carbon metabolism. Significance: The study identifies chloroplastic aldolases as new lysine-methylated proteins.

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confidence: 99%

Characterization of Chloroplastic Fructose 1,6-Bisphosphate Aldolases as Lysine-methylated Proteins in Plants

Mininno

Brugière

Pautre

et al. 2012

“…pET21a TbPRMT7 and pET28b human PRMT6 were purified as PRMT1. pET28a human PRMT3 (residues 211-531) was grown and purified as described in Wang et al (24 (26). Time-dependent incorporation of the radiolabel into substrates was quantified using a scintillation counter (Beckman Coulter).…”

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confidence: 99%

Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity

Morales

Nitzel

Price

et al. 2015

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“…Radiolabel incorporation over time was measured using the discontinuous ZipTip assay described previously. Briefly, 10-l reaction samples of different time points were taken out and quenched by 6 M guanidine HCl solution and processed with ZipTip C18 assay to quantitate the amount of methylated product (21). The resulting time course of [ 3 H]AdoMet incorporation was fit into single exponential curve, y ϭ a⅐(1 Ϫexp(Ϫb⅐x)), to determine the parameters a (maximum product concentration) and b (k chem ).…”

Section: Methodsmentioning

confidence: 99%

“…The same kinetic assay was employed to measure the activity of these three mutants with the R3 peptide; however, the turnover rate was very slow and beyond the limit of detection for this method (data not shown). We turned to a more sensitive method that we recently reported on (21) and examined the mutant activity on a protein substrate, hnRNP K (supplemental Table S4). With all larger sized mutations, the activity was severely impaired.…”

Section: Analysis Of the Products Formed By M48a-and M48l-prmt1-previousmentioning

confidence: 99%

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Investigation of the Molecular Origins of Protein-arginine Methyltransferase I (PRMT1) Product Specificity Reveals a Role for Two Conserved Methionine Residues

Gui

Wooderchak

Daly

et al. 2011

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Protein-arginine methyltransferases aid in the regulation of many biological processes by methylating specific arginyl groups within targeted proteins. The varied nature of the response to methylation is due in part to the diverse product specificity displayed by the protein-arginine methyltransferases. In addition to site location within a protein, biological response is also determined by the degree (mono-/dimethylation) and type of arginine dimethylation (asymmetric/symmetric). Here, we have identified two strictly conserved methionine residues in the PRMT1 active site that are not only important for activity but also control substrate specificity. Mutation of Met-155 or Met-48 results in a loss in activity and a change in distribution of mono-and dimethylated products. The altered substrate specificity of M155A and M48L mutants is also evidenced by automethylation. Investigation into the mechanistic basis of altered substrate recognition led us to consider each methyl transfer step separately. Single turnover experiments reveal that the rate of transfer of the second methyl group is much slower than transfer of the first methyl group in M48L, especially for arginine residues located in the center of the peptide substrate where turnover of the monomethylated species is negligible. Thus, altered product specificity in M48L originates from the differential effect of the mutation on the two rates. Characterization of the two active-site methionines provides the first insight into how the PRMT1 active site is engineered to control product specificity.Protein methylation is a significant post-translational modification in eukaryotic organisms. Protein arginine residues can be methylated on the guanidino nitrogens by protein-arginine methyltransferases (PRMTs), 2 which use S-adenosyl-L-methionine (AdoMet) as a methyl group donor. This post-translational modification is important in a wide variety of fundamental biological processes, including transcription, RNA splicing, signal transduction, DNA repair, viral replication (reviewed in Ref. 1), and chromatin remodeling (2). In recent years, the significance of PRMTs in human diseases has been increasingly studied, especially in cardiovascular disease (3) and cancer (4). In all, PRMTs play a crucial role in many biological processes.Although the biological importance of PRMTs has become well accepted, the current knowledge of the fundamental biochemistry of these enzymes is limited, due in part to the complexity of the system. So far, 11 PRMT isoforms have been identified. In mammalian cells, nine PRMTs catalyze monomethyl arginine (MMA) formation, and they can be categorized into two major types as follows: PRMT1, -2-4, -6, and -8 additionally catalyze asymmetric dimethyl arginine (ADMA) formation, demonstrating type I activity; and PRMT5, -7, and -9 catalyze symmetric dimethyl arginine (SDMA) formation, demonstrating type II activity (Fig. 1A). PRMT10 and -11 were identified as putative PRMT genes with no methylation activity shown as yet (5). As with other enzyme f...