Affinity labelling of rat-muscle hexokinase type II by a glucose-derived alkylating agent.
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 alkylating 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 x 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.
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D005944:Glucosamine / Q000031:analogs & derivatives*; D006593:Hexokinase / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009132:Muscles / Q000201:enzymology*; D051381:Rats
D000345:Affinity Labels; C018852:N-bromoacetylglucosamine; D006593:Hexokinase; D005944:Glucosamine; D000117:Acetylglucosamine