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 ∼3 Å 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. Structural and kinetic studies were done with β-galactosidases with substitutions for Asn460. The substituted enzymes have enhanced substrate affinity in the shallow site indicating lower E·substrate complex energy levels. They have poor transition state stabilization in the deep site that is manifested by increased energy levels of the E·transition state complexes. These changes in stability result in increased activation energies and lower kcat values. Substrate affinity to N460D-β-galactosidase was enhanced through greater binding enthalpy (stronger H-bonds through the bridging water) while better affinity to N460T-β-galactosidase occurred because of greater binding entropy. The transition states are less stable with N460S- and N460T-β-galactosidase because of the weakening or loss of the important bond to the O2 hydroxyl of the transition state. For N460D-β-galactosidase, the transition state is less stable due to an increased entropy penalty.

► The activity of β-galactosidase decreases substantially upon substituting for Asn460. ► Binding of O3 hydroxyl of galactose of the substrate via water is good or better. ► Binding O2 hydroxyl of transition state analogs is poor and kcat/Km values decrease. ► Studies show why substrate binding and transition state stabilization are altered. ► Better substrate binding and poorer transition state stability cause activity decreases.