1. Substrate-saturation curves of brain hexokinase for MgATP2− were sigmoidal at sub-saturating concentrations of glucose when the Mg2+/ATP ratio was maintained at 1:1. Under identical conditions, except that Mg2+ was present in excess, hyperbolic curves were observed. 2. The number of binding sites (calculated from Hill plots) is 1.8 at a Mg2+/ATP ratio 1:1, and 1.0 with excess of Mg2+. The apparent Km for MgATP2− is 6.5×10−4m at a Mg2+/ATP ratio 1:1, and 3.5×10−4m with excess of Mg2+. 3. Interdependence between substrate-binding sites was indicated by the effects of varying the concentration of glucose. The sigmoidality and deviation from Michaelis–Menten kinetics at a Mg2+/ATP ratio 1:1 became less pronounced with increasing glucose concentration. Also, although substrate-saturation curves for glucose were hyperbolic when the Mg2+/ATP ratio was 1:1, reciprocal plots were non-linear. These were linear with excess of Mg2+. 4. High concentrations of Mg2+ (Mg2+/ATP ratios above 5:1) were inhibitory. 5. The results are taken to indicate homotropic co-operative binding of MgATP2− and that Mg2+ is an allosteric activator. Possible implications in regulation are discussed.

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