Inhibition of human milk bile-salt-dependent lipase by boronic acids. Implication to the bile salts activator effect

Abstract

The bile-salt-dependent lipase from human milk, which catalyzes the hydrolysis of the water-soluble substrate 4-nitrophenyl acetate and the water-insoluble substrate tributyrin, is competitively inhibited by phenyl boronic acid. This inhibitor does not interfere with the interaction of lipase either with the siliconized glass beads/water interface or with the activator bile-salt binding site. The boronic acid binds near or at the active site serine, since modification of this residue by diisopropylphosphofluoridate (DFP) was prevented by phenyl boronic acid. Phenyl boronic acid binds 15-fold as tightly to bile-salt-dependent lipase as does 4-nitrophenyl acetate. Therefore, phenyl boronic acid bears analogy to a substrate rather than to a tetrahedral intermediate analog. Bile salts such as sodium taurocholate which are non-essential activators for the milk lipase activity on water-soluble substrates decrease the Km as well as the enzyme inhibitor dissociation constant (Ki). They have a slight effect on kcat. These results are interpreted in terms of an increase of the stability of the enzyme-substrate tetrahedral intermediate and in general of any transition states for the formation and for the decomposition of these intermediates upon the enzyme bile salts interaction.