Reaction of rat hepatic glucokinase with substrate-related and other alkylating agents

• Published in: European journal of biochemistry Vol. 99; no. 2; pp. 299 - 308
• Main Authors: Connolly, B.A, Trayer, I.P. (Birmingham Univ. (UK). Dept. of Biochemistry)
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.09.1979
• Subjects: Acetylglucosamine - analogs & derivatives; Acetylglucosamine - chemical synthesis; Affinity Labels; Alkylating Agents - metabolism; Alkylation; Animals; Glucokinase - antagonists & inhibitors; Hydrogen-Ion Concentration; Kinetics; Liver - enzymology; Rats
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1979.tb13257.x

The reaction of rat hepatic glucokinase with a variety of alkylating agents has been 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. However, whereas N‐bromoacetylglucosamine is a true affinity label for rat muscle hexokinase type II [Connolly and Trayer (1979) Eur. J. Biochem. 93, 375–385] 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 three‐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 are in accord 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. Thus it would seem that the modified residue is not a cysteine unless it is unusually perturbed. This behaviour 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.