REACTION OF RAT HEPATIC GLUCOKINASE WITH SUBSTRATE-RELATED AND OTHER ALKYLATING-AGENTS

The reaction of rat hepatic glucokinase with a variety of alkylating agents was investigated. The substrate-related alkylating agents N-bromoacetylglucosamine, N-(N-bromoacetyl)glycylglucosamine and N-(N-bromoacetyl)-6-aminohexanoylglucosamine are all good competitive inhibitors, with respect to glucose, of the glucokinase reaction. Whereas N-bromoacetylglucosamine is a true affinity label for rat muscle hexokinase type II, it inactivates the liver enzyme only very slowly. Time-dependent irreversible inactivation of glucokinase is best achieved when the bromoacetyl group is made more mobile by interposing at least a 3-atom spacer molecule between it and the sugar ring. The substrate-unrelated but chemically similar reagents, N-bromoacetylgalactosamine and bromoacetic acid, do not inhibit glucokinase and only cause slight non-specific inactivation. The kinetics of inactivation of glucokinase by N-(N-bromoacetyl)-6-aminohexanoylglucosamine agree with the reversible formation of an enzyme-inhibitor complex prior to modification, indicating that the reagent is active site directed. Both glucose and N-acetylglucosamine protect competitively against inactivation. Use of 14C-labelled reagent showed that 1 mol reagent/mol protein caused complete activity loss, although an additional 1 mol/mol protein reacted without any effect on activity. The inactivation rate is relatively insensitive to pH variation over the range 6.5-9. The modified residue may not be cysteine unless it is unusually perturbed. This behavior contrasts with that of yeast hexokinase, bovine brain hexokinase type I and rat muscle hexokinase type II with similar affinity reagents where a thiol is modified with complete activity loss.