In "E. coli", Glu-461 was thought to be the nucleophile in the substitution reaction. However, it is now known that Glu-461 is an acid catalyst. Instead, Glu-537 is the actual nucleophile, binding to a galactosyl intermediate. In humans, the nucleophile of the hydrolysis reaction is Glu-268. Gly794 is important for β-gal activity. It is responsible for putting the enzyme in a "closed", ligand bounded, conformation or "open" conformation, acting like a "hinge" for the active site loop. The different conformations ensure that only preferential binding occurs in the active site. In the presence of a slow substrate, Gly794 activity increased as well as an increase in galactosylation and decrease in degalactosylation.