Donor‐induced conformational gating and substrate‐assisted catalysis in α ‐1,3‐galactosyltransferase

Abstract

Retaining glycosyltransferases catalyze the formation of stereochemically conserved glycosidic bonds through mechanisms that remain debated. Using bovine α 1,3‐galactosyltransferase ( α 3GalT) as a model, we combine mutagenesis, equilibrium unfolding, kinetics, and molecular dynamics simulations to understand how donor‐induced loop ordering promotes catalysis. Alanine‐scanning mutagenesis of the C‐terminal loop (Thr358‐Val368) identified Lys359, Tyr361, and Arg365 as critical for donor binding, catalysis, and ligand‐dependent stabilization. In addition, D225A and E317A were inactive and showed minimal ligand‐induced stabilization, consistent with impaired metal binding and substrate stabilization, respectively. Donor binding induces an ordered conformation in the C‐terminus, reducing its local flexibility by 30% and pre‐organizing the active site for catalysis. MD‐derived energy profiles differed markedly for the donor (UDP‐Gal) and acceptor (lactose) in the ternary complex. In this context, experimental apparent K ₘ values indicate higher donor affinity than acceptor affinity. Our results show that donor binding stabilizes the C‐terminal loop, assembling a competent complex for catalysis. These findings support a general coupling between conformational gating, donor stabilization, and the catalytic mechanism in retaining GT‐A‐fold enzymes.