Zinc-Homocysteine Binding in Cobalamin-Dependent Methionine Synthase and its Role in the Substrate Activation: DFT, ONIOM, and QM/MM Molecular Dynamics Studies

Cobalamin-dependent methionine synthase (MetH) is an important metalloenzyme responsible for the biosynthesis of methionine. It catalyzes methyl transfer from N-5-methyltetrahydrofolate to homocysteine (Hcy) by using a zinc ion to activate the Hcy substrate. Density functional theory (B3LYP) calculations on the active-site model in gas phase and in a polarized continuum model were performed to study the Zn coordination changes from the substrate-unbound state to the substrate-bound state. The protein effect on the Zn2+ coordination exchange was further investigated by ONIOM (B3LYP:AMBER)-ME and EE calculations. The Zn2+-coordination exchange is found to be highly unfavorable in the gas phase with a high barrier and endothermicity. In the water solution, the reaction becomes exothermic and the reaction barrier is drastically decreased to about 10.0 kcal/mol. A considerable protein effect on the coordination exchange was also found; the reaction is even more exothermic and occurs without barrier. The enzyme was suggested to constrain the zinc coordination sphere in the reactant state (Hcy-unbound state) more than that in the product state (Hcy-bound state), which promotes ligation of the Hcy substrate. Molecular dynamics simulations using molecular mechanics (MM) and PM3/MM potentials suggest a correlation between the flexibility of the Zn2+-binding site and regulation of the enzyme function. Directed in silico mutations of selected residues in the active site were also performed. Our studies support a dissociative mechanism starting with the Zn-O-(Asn234) bond breaking followed by the Zn-S-(Hcy) bond formation; the proposed associative mechanism for the Zn2+-coordination exchange is not supported. (C) 2011 Wiley Periodicals, Inc. J Comput Chem 32: 3154-3167, 2011