The role of magnesium ions in β-galactosidase-catalysed hydrolyses. Studies on charge and shape of the β-galactopyranosyl-binding site

Abstract

1. β-d-Galactopyranosyl trimethylammonium bromide is a competitive inhibitor of β-galactosidase, Ki=1.4±0.2mm at 25°C. 2. Tetramethylammonium bromide is not an inhibitor (Ki>0.2m). 3. The kinetics of deactivation of Mg2+-saturated, and of inhibitor-and Mg2+-saturated, enzyme in 10mm-EDTA are similar. 4. The apparent Ki for the glycosylammonium salt is approx. 2.2mm in the absence of Mg2+. 5. It is therefore concluded that Mg2+ and the inhibitor bind independently, and that the Mg2+ does not act as an electrophilic catalyst. 6. Complexant fluorescence measurements indicate binding of 1 Mg2+ ion per 135000-dalton protomer. 7. This stoicheiometry is confirmed by equilibrium dialysis. 8. 1,6-Anhydrogalactopyranose is neither a substrate (kcat./Km< 3×10-2m-1·S-1) nor an inhibitor (Ki>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.