Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase. Sequence homology with triosephosphate isomerase.
N-Bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pKa values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo[14C2]acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivty methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene.
D000345:Affinity Labels / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000037:antagonists & inhibitors*; D004983:Ethanolamines / Q000494:pharmacology*; D005260:Female; D005956:Glucose-6-Phosphate Isomerase / Q000037:antagonists & inhibitors*; D006639:Histidine / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D011487:Protein Conformation; D011817:Rabbits; D014305:Triose-Phosphate Isomerase / Q000037:antagonists & inhibitors*
D000345:Affinity Labels; D000596:Amino Acids; D004983:Ethanolamines; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D006639:Histidine; C020161:N-bromoacetylethanolamine phosphate; D002238:Carbohydrate Epimerases; D014305:Triose-Phosphate Isomerase; D005956:Glucose-6-Phosphate Isomerase