Sodium-dependent activation of intestinal brush-border sucrase: Correlation with activation by deprotonation from pH 5 to 7

The activation of rabbit intestinal brush-border sucrase in the pH range 4.8 to 9.2 was studied as a function of sucrose concentration and temperature in a metal-free, n-butylamine universal buffer, both in the absence and in the presence of sodium. When sodium was absent, enzyme activation involved the simultaneous loss of two key protons (pK₁ of about 5.6), thus yielding a high-affinity, catalytically active enzyme conformation. Inactivation followed when a third key proton (pK₂ of about 8.4) was lost. When sodium was present, kinetic analysis in the pH range 4.8 to 7.2 revealed that sodium activation involves distinct effects on the two kinetic parameters, V_m and K_m. The V_m parameter seemed to conform to the classical rules of pH-dependent enzyme activation and implicated the release of a single proton whose apparent pK (pK_1y, about 5.6) was little affected by sodium. On the contrary, the K_m parameter was strongly influenced by sodium. Here, activation of rabbit sucrase seemed to involve release of a different proton whose apparent pK (pK_1x also of about 5.6 in the absence of sodium) was strongly shifted to more acid values by saturating sodium concentrations. The functional distinction between the above two protons explains the existence of strong affinity-type activating effects of sodium on rabbit sucrase, previously shown to be pH independent (F. Alvarado and A. Mahmood, 1979, J. Biol. Chem.254, 9534–9541).