The role of magnesium ions in beta-galactosidase hydrolyses. Studies on charge and shape of the beta-galactopyranosyl binding site.
1. beta-d-Galactopyranosyl trimethylammonium bromide is a competitive inhibitor of beta-galactosidase, K(i)=1.4+/-0.2mm at 25 degrees C. 2. Tetramethylammonium bromide is not an inhibitor (K(i)>0.2m). 3. The kinetics of deactivation of Mg(2+)-saturated, and of inhibitor-and Mg(2+)-saturated, enzyme in 10mm-EDTA are similar. 4. The apparent K(i) for the glycosylammonium salt is approx. 2.2mm in the absence of Mg(2+). 5. It is therefore concluded that Mg(2+) and the inhibitor bind independently, and that the Mg(2+) does not act as an electrophilic catalyst. 6. Complexant fluorescence measurements indicate binding of 1 Mg(2+) ion per 135000-dalton protomer. 7. This stoicheiometry is confirmed by equilibrium dialysis. 8. 1,6-Anhydrogalactopyranose is neither a substrate (k(cat.)/K(m)< 3x10(-2)m(-1).S(-1)) nor an inhibitor (K(i)>0.2m). 9. Considerations of conformations available to the cationic inhibitor and to the anhydrogalactose indicate that the substrate is bound with the pyranose ring in a conformation not greatly different from the normal chair (C1) conformation.
D001665:Binding Sites; D001966:Bromine; D003956:Dialysis; D005690:Galactose; D005696:Galactosidases / Q000037:antagonists & inhibitors; D007700:Kinetics; D008274:Magnesium; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D011714:Pyrans; D000644:Quaternary Ammonium Compounds; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D011714:Pyrans; D000644:Quaternary Ammonium Compounds; D005696:Galactosidases; D008274:Magnesium; D001966:Bromine; D005690:Galactose