A theoretical study on the catalytic mechanism of the retaining α-1,2-mannosyltransferase Kre2p/Mnt1p: the impact of different metal ions on catalysis

Glycosyltransferases are sugar-processing enzymes that require a specific metal ion cofactor for catalysis. In the presence of other ions the catalysis is often impaired. Here, for the first time, the enzymatic catalysis in the presence of various metal ions was modeled for a glycosyltransferase using a large enzymatic model. The catalytic mechanism of alpha-1,2-mannosyltransferase Kre2p/Mnt1p in the presence of Mn2+ and other ions (Mg2+, Zn2+ and Ca2+) was modeled at the two hybrid DFT-QM/MM (M06-2X/OPLS2005 and B3LYP/OPLS2005) levels. Kinetic and structural parameters of transition states and intermediates, as well as kinetic isotope effects, were predicted and compared with available experimental and theoretical data. The catalysis in the presence of the metal ions is predicted as a stepwise S(N)i-like nucleophilic substitution reaction (D-Nint*A(N)(double dagger)D(h)A(xh)) via oxocarbenium ion intermediates. In the rate-determining step the leaving phosphate group of the donor substrate plays a role of the base catalyst. The predicted increased enzymatic reactivity (k(cat): Zn2+ approximate to Mg2+ < Mn2+ < Ca2+) correlated with the metal ion ability to polarize the Kre2p environment (Mg2+ > Zn2+ > Mn2+ > Ca2+). The formation of the retained anomeric configuration in the product is controlled by a strict geometry of the active site of Kre2p. The 6-OH group of the attacking acceptor substrate may assist in protection of the anomeric carbon against unwanted hydrolysis by a through-space interaction with the electron deficient C1 = O5(+) moiety of the oxocarbenium-ion-like transition state.