Significance Recent exome sequencing studies have uncovered high-frequency histone H3 driver mutations in pediatric cancers. Previous studies have shown that lysine to methionine histone mutations are potent inhibitors of their respective lysine methyltransferases. However, an in-depth understanding of this inhibition was limited by the lack of structural and kinetic information. This study investigates the biochemical and biophysical parameters of lysine to methionine histone mutants using the methyltransferase G9a and H3K9M as a model system. Structural and functional experiments conclude that the methyltransferase cofactor S -adenosyl methionine is required for binding of G9a to the mutant histone.