Cascading proton transfers are a hallmark of the catalytic mechanism of SAM-dependent methyltransferases.

: The S-adenosyl-L-methionine (SAM)-dependent methyltransferases attach a methyl group to the deprotonated methyl lysine (Kme0) using SAM as a donor. An intriguing, yet unanswered, question is how the deprotonation of the methyl lysine takes place which results in a lone pair of electrons at the Nϵ atom of the methyl lysine for the following methyl transfer. PRDM9, one of the few methyltransferases with well-defined enzyme activity in vitro and in vivo, is a good representative of the PR/SET domain methyltransferase family to study the deprotonation and subsequently the methyl transfer. The reaction consists of two progressing steps: (i) the absolutely required substrate methyl lysine deprotonation and (ii) the transfer of the methyl group to the deprontonated methyl lysine. We use empirical valence bond (EVB) simulations to evaluate Y357 at the active site as potential general base for the deprotonation of the methyl lysine. Indeed, our study has found that the pKa of Tyr357 is low enough to make it an ideal candidate for proton abstraction from the methyl lysine. The partially deprontonated Tyr357 is able to change its H-bond pattern thus bridging two proton tunneling states (OH- H 0-Tyr357 and Kme0-Nϵ H O-Tyr357) and providing a cascading proton transfer from Tyr357 to hydroxide, generating deprotonated Tyr357 and then from Kme0 to the deprotonated Tyr357 resulting in deprotonated methyl lysine. This cascading proton transfer shortens the lifespan of the labile intermediates, and affects the conformational changes during the product release important to promote the proton release to the bulk solvent. Our computational efforts have uncovered a new catalytic mechanism to unravel the unanswered question about the deprotonation of the methyl lysine in methyltransferases.