Catalytic Mechanism and Product Specificity of Rubisco Large Subunit Methyltransferase: QM/MM and MD Investigations

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Abstract
Molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations have been carried out in an investigation of Rubisco large subunit methyltransferase (LSMT). It was found that the appearance of a water channel is required for the stepwise methylation by S-adenosylmethionine (AdoMet). The water channel appears in the presence of AdoMet (LSMT·Lys-NH3+·AdoMet), but is not present immediately after methyl transfer (LSMT·Lys-N(Me)H2+·AdoHcy). The water channel allows proton dissociation from both LSMT·AdoMet·Lys-NH3+ and LSMT·AdoMet·Lys-N(Me)H2+. The water channel does not appear for proton dissociation from LSMT·AdoMet·Lys-N(Me)2H+, and a third methyl transfer does not occur. By QM/MM, the calculated free energy barrier of the first methyl transfer reaction catalyzed by LSMT (Lys-NH2 + AdoMet → Lys-N(Me)H2+ + AdoHcy) is ΔG‡ = 22.8 ± 3.3 kcal/mol. This ΔG‡ is in remarkable agreement with the value 23.0 kcal/mol calculated from the experimental rate constant (6.2 × 10-5 s-1). The calculated ΔG‡ of the second methyl transfer reaction (AdoMet + Lys-N(Me)H → AdoHcy + Lys-N(Me)2H+) at the QM/MM level is 20.5 ± 3.6 kcal/mol, which is in agreement with the value 22.0 kcal/mol calculated from the experimental rate constant (2.5 × 10-4 s-1). The third methyl transfer (Lys-N(Me)2 + AdoMet → Lys-N(Me)3+ + AdoHcy) is associated with an allowed ΔG‡ of 25.9 ± 3.2 kcal/mol. However, this reaction does not occur because a water channel does not form to allow the proton dissociation of Lys-N(Me)2H+. Future studies will determine whether the product specificity of lysine (mono, di, and tri) methyltransferases is determined by the formation of water channels.