SET7/9 exhibits sigmoidal kinetics on nucleosomes, hyperbolic kinetics on histones by an ordered sequential mechanism, and methylates lysine and arginine

SET7/9 (SETD7) is a SET domain protein lysine methyltransferase (PKMT). We characterized its activity using a mass spectrometry (MS) assay, showing that it follows an ordered sequential enzyme kinetic mechanism where SAM is the first substrate to bind, followed by histone H3, and mono-methylated histone H3 is the first product to dissociate, followed by SAH. Full-length histones H2A, H2B, and H4 are also substrates for SET7/9. We found that SET7/9 methylates nucleosome core particles (NCP), histone H2BK120 ubiquitinated nucleosome core particles (Ub-NCP) as well as histone octamers, exhibiting sigmoidal kinetics, and suggesting an allosteric interaction that was not observed with free histones, which follow hyperbolic (Michaelis-Menten) kinetics. Using low (25 nM) SET7/9, we only detect monomethyl-lysine with all substrates except Ub-NCP, which produces mono- and di-methyllysine at equal rates. Proteomic analysis shows that SET7/9 catalyzes multiple sites and types of, methylation on histones, depending on if they are free histones, or within an octamer. Free histone H3 is methylated at multiple N-terminal sites (including H3K4) that are not methylated in the octamer. Some C-terminal methylation sites were discovered that can also be ubiquitinated including H2BK120. Since lysine methylation and ubiquitination are mutually exclusive such methylation by SET7/9 may serve to prevent ubiquitination. At high enzyme and substrate concentrations and extended incubation times, we show that SET7/9 can catalyze the formation of dimethyl- and trimethyllysine on H2B, H3 and histone octamers and, most remarkably, the formation of monomethyl- and dimethylarginine on histone H3 and within octamers.