of substrate and cofactor allows SAM to dissociate without disrupting substrate binding and can lead to multiple rounds of lysine methylation without substrate dissociation. Although neither a substrate-bound or SAM-bound crystal structure for EZH2 has been determined, STAMP structure alignment with the human SET7/9 methyltransferase shows conserved tyrosine residues in almost identical positions within the putative active site of EZH2. It had been previously suggested that tyrosine 726 in the EZH2 active site was acting as a general base to de-protonate the substrate lysine but kinetic isotope effects have indicated that active site residues are not directly involved in the chemistry of the methyltransferase reaction. Instead these experiments support a mechanism in which the residues lower the pKa of the substrate lysine residue while simultaneously providing a channel for water to access the lysine side chain within the interior of the active site. Bulk solvent water can then easily deprotonate the lysine side chain, activating it for nucleophilic attack of the SAM cofactor in an SN2-like reaction resulting in transfer of the methyl group from SAM to the lysine side chain. EZH2 primarily catalyzes mono- and di-methylation of H3K27 but a clinically relevant mutation of residue tyrosine 641 to phenylalanine (Y641F) results in higher H3K27 tri-methylation activity. It is proposed that the removal of the hydroxyl group on Y641 abrogates steric hindrance and allows for accommodation of a third methyl group on the substrate lysine. Clinical significance Cancer EZH2 is an attractive target for anti-cancer therapy because it helps cancerous cells divide and proliferate. It is found in larger amounts than in healthy cells in a wide range of cancers including breast, prostate, bladder, uterine, and renal cancers, as well as melanoma and lymphoma. EZH2 is a gene suppressor, so when it becomes overexpressed, many tumor suppressor genes that are normally turned on, are turned off. Inhibition of EZH2 function shrinks malignant tumors in some reported cases because those tumor suppressor genes are not silenced by EZH2. EZH2 typically is not expressed in healthy adults; it is only found in actively dividing cells, like those active during fetal development. Because of this characteristic, overexpression of EZH2 can be used as a diagnostic marker of cancer and some neurodegenerative disorders. However, there are cases where it is difficult to tell whether overexpression of EZH2 is the cause of a disease, or simply a consequence. If it is only a consequence, targeting