Affinity Labelling of Rat‐Muscle Hexokinase Type II by a Glucose‐Derived Alkylating Agent

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Abstract
The glucose‐derived alkylating agent N‐bromoacetylglucosamine (GlcNBrAc) is shown to cause a time‐dependent irreversible inactivation of rat muscle hexokinase type II. The kinetics of inactivation are in accord with the reversible formation of an enzyme‐inhibitor complex prior to modification, indicating that the reagent is active‐site‐directed. A Ki of 0.57 mM obtained for this reversible complexing is in agreement with a Ki of 0.65 mM obtained for the inhibition caused by N‐propionylglucosamine, an isosteric analogue of GlcNBrAc and a competitive inhibitor with respect to glucose. Glucose itself protects competitively against inactivation. A KG of 0.26 mM obtained for the formation of enzyme‐glucose complex from these studies is in agreement with the kinetically‐determined Km of 0.2 mM. The substrate‐unrelated but chemically similar alkylatig agents bromoacetic acid and N‐bromoacetylgalactosamine inactivate the enzyme at 20% of the rate caused by GlcNBrAc. The inactivation rate increases rapidly over the pH range 7–9. Analysis of this pH dependence shows that a single residue of pKa 8.9 is reacting with GlcNBrAc with a kmax (pH corrected, pseudo‐first‐order rate constant) of 1.5 × 10−3 s−1. These values are typical of the reaction of model thiols with alkylating agents and suggests the reacting residue is probably a cysteine. Use of radioactively labelled GlcNBrAc indicates that uptake of 1 mol of reagent per mol protein causes complete activity loss. Finally the behaviour of this enzyme with active‐site‐directed alkylating agents is compared with published results of similar experiments carried out with yeast hexokinase and bovine brain hexokinase type I.