Computational Study of Mycobacterium smegmatis Acyl Transferase Reaction Mechanism and Specificity
ACS Catal. 2018, 8, 10698–10706.
The acyl transferase from Mycobacterium smegmatis (MsAcT) catalyzes the acyl transfer between a range of primary and secondary alcohols, whereby its outstanding ability is to perform this reaction in aqueous solution. Therefore, MsAcT opens diﬀerent options for acylation reactions enabling alternatives for many conventionally hydrolytic enzymes used in biocatalysis. Nevertheless, hydrolysis is still a major side reaction of this enzyme. To provide a detailed understanding of the competition between hydrolysis and transesteriﬁcation reactions, a combination of density functional theory and free energy perturbation methods have been employed. The relative binding free energies and the energy proﬁles of the chemical steps involved in the reaction were calculated for a number of substrates. The calculations show that the enzyme active site exhibits a higher aﬃnity for substrates with an aromatic ring. The rate-determining step corresponds to the collapse of a negatively charged tetrahedral intermediate in the substrate acylation half-reaction. The intrinsic barriers of the transesteriﬁcation and hydrolysis half-reactions are calculated to be of similar heights, suggesting that the determining factor in the MsAcT speciﬁcity is the higher binding aﬃnity of the active site for the alcohol substrates relative to water. Finally, the inﬂuence of the acyl donor on the MsAcT-catalyzed reaction is also investigated by considering diﬀerent esters in the calculations.