Substitution for Asn460 cripples β-galactosidase (Escherichia coli) by increasing substrate affinity and decreasing transition state stability.

Substrate initially binds to β-galactosidase (Escherichia coli) at a 'shallow' site. It then moves ∼3Å to a 'deep' site and the transition state forms. Asn460 interacts in both sites, forming a water bridge interaction with the O3 hydroxyl of the galactosyl moiety in the shallow site and a direct H-bond with the O2 hydroxyl of the transition state in the deep site.