AbstractProtein lysine methyltransferases have important regulatory functions in cells, but mechanisms determining their activity and specificity are incompletely understood. Naturally, SETD2 introduces H3K36me3, but previously an artificial super-substrate (ssK36) was identified, which is methylated >100-fold faster. The ssK36-SETD2 complex structure cannot fully explain this effect. We applied molecular dynamics (MD) simulations and biochemical experiments to unravel the mechanistic basis of the increased methylation of ssK36, considering peptide conformations in solution, association of peptide and enzyme, and formation of transition-state (TS) like conformations of the enzyme-peptide complex. We observed in MD and FRET experiments that ssK36 adopts a hairpin conformation in solution with V35 and K36 placed in the loop. The hairpin conformation has easier access into the active site of SETD2 and it unfolds during the association process. Peptide methylation experiments revealed that introducing a stable hairpin conformation in the H3K36 peptide increased its methylation by SETD2. In MD simulations of enzyme-peptide complexes, the ssK36 peptide approached TS-like structures more frequently than H3K36 and distinct, substrate-specific TS-like structures were observed. Hairpin association, hairpin unfolding during association, and substrate-specific catalytically competent conformations may also be relevant for other PKMTs and hairpins could represent a promising starting point for SETD2 inhibitor development.