Determining the kinetic parameters of the protein lysine methyltransferase SET7/9 using liquid chromatography and tandem mass spectrometry

Abstract

Histone lysine methylation is an epigenetic post-translational modification which can modulate gene expression and has been implicated in various forms of cancer. The aim of this research is to determine the kinetic parameters of the lysine methyltransferase SET7/9 using liquid-chromatography and tandem mass spectrometry (LC-MS/MS) techniques. Reactions are performed in vitro using SET7/9 enzyme, S-adenosyl-L-methionine (SAM), and recombinant histone H3 or a peptide of the N-terminal tail of histone H3. Analysis is performed using two assays, one to quantify histone modifications (especially mono-, di-, and trimethyl lysine) and one to quantify S-adenosyl-L-homocysteine (SAH), a co-product of all SAM-dependent methyltransferase reactions. The data collected are then used to calculate the apparent kinetic parameters of the reaction, Kmapp and Vmaxapp. For SET7/9 the Kmapp and Vmaxapp for SAM were 2.24 ± 0.97 µM and 0.047 ± 0.0057 pmol/min when full-length histone H3 was used, and 0.22 ± 0.03 µM and 0.19 ± 0.004 pmol/min when H3 peptide was used; the Kmapp and Vmaxapp for histone H3 were 1.21 ± 0.53 µM and 0.16 ± 0.018 pmol/min.