Studies on hexokinase: 1. The hexokinase activity of rat-brain extracts

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Abstract
The reactions occurring in aqueous rat-brain extracts during the phosphorylation of glucose by adenosinetriphosphate (ATP) and the properties of rat-brain hexokinase were investigated. Estimation of the Michaelis constant yielded approx. values of 5 x 10-5[image] for hexokinase and 7 x 10-4 [image]M for phosphohexokinase. The affinity for ATP was the same for both enzymes. The Michaelis constant in this case was about 8 x 10-4[image]. A study of the reaction products showed that glucose was quantitatively converted to hexosediphosphate and that glycolysis did not, under the conditions used, proceed beyond the triosephosphate stage. In the initial stages of the reaction some hexosemonophosphate might accumulate, especially with diluted enzyme. The brain extracts used contained somewhat variable amts. of apyrase, myokinase and adenylic deaminase. The activity of myokinase was sufficient to prevent the accumulation of any but small amts. of adenosinediphosphate (ADP). The reaction rate was about 30% slower as phosphate donor than in the presence of ATP. But since ATP was always present in excess, even at the end of expts., the activity of myokinase was never a limiting factor in the reaction and was sufficient to prevent the accumulation of any but small amts. of ADP. The ammonia formation from ATP, in absence of phosphate acceptors, was limited by the activity of apyrase rather than adenylic deaminase, while the ammonia formation from ADP was limited by the activity of adenylic deaminase rather than myokinase. Brain hexokinase was strongly inhibited by hexosemonophosphates. Glucose-6-phosphate was found to be more inhibitory than fructose-6-phosphate, while Robison ester had an effect intermediate between the 2 components. The results suggested that the inhibitory compound was glucose-6-phosphate. The inhibition by hexosemonophosphates was non-competitive with respect either to glucose or ATP. No summation of initial reaction rates of hexokinase and phosphohexokinase was observed when glucose and hexosemonophosphate were simultaneously added to brain extract, because hexokinase activity was inhibited in presence of hexosemonophosphate. The addition of various substances known to have protective or activating effects on enzymes in general and on hexokinase in particular, alone or in combination, had no effect on the hexokinase activity of rat-brain extracts.
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