Reaction of brain hexokinase with a substrate-like reagent. Alkylation of a single thiol at the active site.
An analogue of the substrate glucose, N-(bromoacetyl)-D-glucosamine (GlcNBrAc) inactivates bovine brain mitochondrial hexokinase completely and irreversibly in a pseudo-first-order fashion at pH 8.5 and 22 degrees C. The rate of inactivation of hexokinase by this reagent does not increase linearly with increasing reagent concentration but exhibits an apparent saturation effect, suggesting the formation of a reversible complex between the enzyme and the reagent prior to the inactivation step. The pH dependence of the rate of inactivation suggests that a group on the enzyme with pKa = 9.1 is being modified by this reagent. At pH 8.0 the rate of inactivation by this reagent is very slow, and it can be shown to be a competitive inhibitor of the hexokinase reaction with respect to the substrate glucose. The substrates glucose and ATP strongly protected the enzyme against the inactivation reaction. The inactivation of the enzyme was found to be accompanied by the alkylation of two sulfhydryl residues as shown by the formation of approximately 2 mol of S-(carboxymethyl)-cysteine/mol of inactivated enzyme. Treatment of the enzyme with 14C-labeled reagent results in the incorporation of approximately 2 mol of reagent/mol of inactivated enzyme. However, the enzyme protected by glucose still shows the incorporation of approximately 1 mol of the labeled reagent/mol of the enzyme. From a tryptic digest of the enzyme inactivated by this reagent, two labeled peptides were obtained, one of which was absent if the labeling reaction was carried out in presence of glucose. These results indicate that the affinity reagent reacts with two thiols, only one of which is crucial for the activity of the enzyme and is located in the region of its active site.
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D005944:Glucosamine / Q000031:analogs & derivatives*; D006593:Hexokinase / Q000378:metabolism*; D007461:Iodoacetates; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000032:analysis; D014357:Trypsin
D000596:Amino Acids; D007461:Iodoacetates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; C018852:N-bromoacetylglucosamine; D006593:Hexokinase; D014357:Trypsin; D005944:Glucosamine; D000117:Acetylglucosamine