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 substituted 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 stabilize the transition state by interacting with the C3 hydroxyl. The substituted enzymes were less stable to heat than was wild-type enzyme (40-90% of the activity was lost in 10 min at 52 degreesC compared to wild-type beta-galactosidase which lost no activity), but the gross physical properties of the substituted enzymes at normal temperatures were not changed. There were also no differences in the ability to bind or to be activated by Mg2+. The substitutions (except Asp) did not affect the pKa for binding substrate in the ground state, but the pKa of the kcat was altered as would be expected for a residue important for binding the transition state. Substitution by Asp may cause a conformational change at high pH values. Activation energy differences (Delta DeltaGS), as determined by differences in kcat/Km values, indicated that substitutions for His-357 caused significant destabilizations of the first transition state (for the step in which the galactoside bond is cleaved and the covalent reaction intermediate is formed). This resulted in decreases of up to 900-fold in k2 for the mononitrophenyl substrates. In contrast, the k3 values (which depend on the energy level of the second transition state) were not decreased as much (<90-fold). In some cases, the k3 values even increased (when Asn was substituted for His-357). The importance of His-357 for stabilization of the transition state was confirmed by studies with transition state analogue inhibitors that showed that His-357 forms strong specific interactions with the C3 hydroxyl of the galactose moiety of the transition state. Studies with substrate analogue inhibitors indicated that His-357 is probably not important for the binding of the substrates themselves.
Division of Biochemistry, Faculty of Science, University of Calgary, Alberta, Canada.
N J
NJ
Roth
B
B
Rob
R E
RE
Huber
D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D017665:Hydroxyl Radical / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009598:Nitrophenylgalactosides / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D009598:Nitrophenylgalactosides; D017665:Hydroxyl Radical; D006639:Histidine; D001616:beta-Galactosidase; D008274:Magnesium