Biological consequences of histone lysine methylation depend on the methylation states of the lysine residues on the tails of histone proteins that are methylated by protein lysine methyltransferases (PKMTs). Therefore, the ability of PKMTs to direct specific degrees of methylation (i.e., product specificity) is an important property for regulation of chromatin structure and gene expression. Here, the free energy simulations based on quantum mechanical/molecular mechanical (QM/MM) potentials are performed for the first, second. and third methyl transfers from S-adenosyl-L-methionine to the epsilon-amino group of the target lysine/methyl lysine in SET8, one of the important PKMTs. The key questions addressed in this paper include the energetic origin of the product specificity and the reasons for the change of he product specificity as a result of the replacement of Tyr334 by Phe. The free energy barriers for the three methyl transfers in SET8 as well as in the mutant obtained from the simulations are found to be well correlated with the experimental observations on the product specificity of SET8 and the change of product specificity as a result of the mutation. The results support the suggestion that the differential free energy barriers for the methyl transfers may determine, at least in part, how the epigenetic marks of lysine methylation are written by the enzymes. Furthermore, the stability of a water molecule to be located at the active site is examined under different conditions using the free energy simulations, and its role in controlling the product specificity is discussed. The QM/MM molecular dynamics (MD) simulations are also performed on the reactant complexes of the first, second, and third methyl transfers. The results show that the information on the ability of the reactant complexes to form the reactive configurations for the methyl transfers may be used as useful indicators in the prediction of product specificity for PKMIs.
