Picomolar inhibition of β-galactosidase (bovine liver) attributed to loop closure.

In an effort to examine similarities in the active sites of glycosidases within the GH35 family, we performed a structure-activity-relationship study using our recently described library of galactonoamidines. The kinetic evaluation based on UV/Vis spectroscopy disclosed inhibition of β-galactosidase (bovine liver) in the picomolar concentration range indicating significantly higher inhibitor affinity than previously determined for β-galactosidase (A. oryzae). Possible alterations in the secondary protein structure or folding were excluded after further examination of the inhibitor binding using CD spectroscopy. Molecular dynamics studies suggested loop closing interactions as a rationale for the disparity of the active sites in the β-galactosidases under investigation.