HIS-357 OF BETA-GALACTOSIDASE (ESCHERICHIA-COLI) INTERACTS WITH THE C3 HYDROXYL IN THE TRANSITION-STATE AND HELPS TO MEDIATE CATALYSIS

The His at position 357 of beta-galactosidase (Escherichia coli) was s ubstituted by an Asp, an Asn, a Leu, and a Phe, and studies done with the substituted enzymes showed that the main role of His-357 is to sta bilize the transition state by interacting with the C3 hydroxyl. The s ubstituted enzymes were less stable to heat than was wild-type enzyme (40-90% of the activity was lost in 10 min at 52 degrees C compared to wild-type beta-galactosidase which lost no activity), but the gross p hysical properties of the substituted enzymes at normal temperatures w ere not changed. There were also no differences in the ability to bind or to be activated by Mg2+. The substitutions (except Asp) did not af fect the pK(a) for binding substrate in the ground state, but the pK(a ) of the k(cat) was altered as would be expected for a residue importa nt for binding the transition state. Substitution by Asp may cause a c onformational change at high pH values. Activation energy differences (Delta Delta G(S)double dagger), as determined by differences in k(cat )/K-m values, indicated that substitutions for His-357 caused signific ant destabilizations of the first transition state (for the step in wh ich the galactoside bond is cleaved and the covalent reaction intermed iate is formed). This resulted in decreases of up to 900-fold in k(2) for the mononitrophenyl substrates. In contrast, the k(3) values (whic h depend on the energy level of the second transition state) were not decreased as much (< 90-fold). In some cases, the k(3) values even inc reased (when Asn was substituted for His-357). The importance of His-3 57 for stabilization of the transition state was confirmed by studies with transition state analogue inhibitors that showed that His-357 for ms strong specific interactions with the C3 hydroxyl of the galactose moiety of the transition state. Studies with substrate analogue inhibi tors indicated that His-357 is probably not important for the binding of the substrates themselves.