Mechanism of the glycosylation step catalyzed by human α-galactosidase: a QM/MM metadynamics study.

The enzyme α-galactosidase (α-GAL), a member of glycoside hydrolase family 27, catalyzes the removal of a nonreducing terminal α-galactose residue from polysaccharides, glycolipids, and glycopeptides. α-GAL is believed to have the double displacement retaining reaction mechanism. In this work, the glycosylation step catalyzed by human α-GAL was computationally simulated with quantum mechanics/molecular mechanics metadynamics. Our simulations show that the overall catalytic mechanism follows a D(N)*A(N)-like mechanism, and the transition state has a oxocarbenium ion like character with a partially formed double bond between the ring oxygen and C5' carbon atoms. In addition, the galactosyl ring of the substrate follows a conformational itinerary of (4)C(1) → E(3)/(4)H(3) → (1)S(3) along the reaction coordinate.