pH kinetic studies of bovine brain hexokinase

The variation of kinetic parameters with pH was examined for bovine brain hexokinase with glucose and MgATP as substrates. The -log V1 and -log (V1/Km) profiles for both substrates were examined and seen to decrease below pH 6.5. All profiles asymptotically approached slopes of -1, indicating that the loss of activity in each instance was due to the protonation of a single group on the enzyme. Analysis of the data indicated two ionizable groups were involved in the reaction. One functions in the binding of ATP and in catalysis while the other participates in the binding of glucose. The -log V1 profiles both showed a "hump" attributed to a loss of activity in the pH region 7.5-5.5. Addition of aluminum ions to the reaction mixture increased the magnitude of the hump, but the inhibition was abolished by the addition of citrate. Kinetic studies carried out at pH 7 indicated that aluminum was a competitive inhibitor with respect to ATP and noncompetitive with respect to glucose. However, secondary plots of the kinetic data were nonlinear, concave downward, indicating that the inhibition is not of a simple type. Possible explanations for this phenomenon are presented.