title: Detection of an essential sulfhydryl group in phosphoglycerate mutase with an affinity-labeling reagent.
abstract: N-Bromoacetylethanolamine phosphate rapidly and irreversibly inactivates rabbit muscle phosphoglycerate mutase. At high molar ratios of reagent to enzyme, loss of activity (both mutase and phosphatase) approximates pseudo-first order kinetics. A rate-saturation effect is observed with half-maximal rate of inactivation occurring at 0.32 mM reagent, a value close to the Km for 3-phosphoglyceric acid. This datum and the dissociation constant of the 2,3-bisphosphoglycerate-enzyme complex, as determined from inactivation kinetics in the presence of the bisphosphate, suggest that the reagent reacts at the substrate binding site. Inactivation results from the covalent incorporation of about 0.8 mol of reagent/mol of catalytic subunit as determined with 14C-labeled reagent. Incorporation is negligible in the presence of substrate and is reduced 8-fold in the presence of 6 M urea. From amino acid analyses on acid hydrolysates of the inactivated enzyme, we have identified a sulfhydryl group as the site of alkylation. A peptide containing the essential sulfhydryl group has been isolated from a tryptic digest of the enzyme inactivated with labeled reagent; its amino acid composition is Trp1, Lys1,-Cys(Cm)1, Asp1, Ser1, Glu2, Gly1, Ala1, Leu1, Phe2.
mesh_terms: D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D004983:Ethanolamines / Q000494:pharmacology; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010736:Phosphoglycerate Mutase / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis
chemical_list: D000345:Affinity Labels; D000596:Amino Acids; D004983:Ethanolamines; D009943:Organophosphorus Compounds; D013438:Sulfhydryl Compounds; C020161:N-bromoacetylethanolamine phosphate; D010770:Phosphotransferases; D010736:Phosphoglycerate Mutase