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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 K/sub m/ 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 /sup 14/C-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 Trp/sub 1/, Lys/sub 1/,Cys(Cm)/sub 1/,Asp/sub 1/,Ser/sub 1/,Glu/sub 2/,Gly/sub 1/,Ala/sub 1/,Leu/sub 1/,Phe/sub 2/.

Authors:
;
Publication Date:
Research Org.:
Oak Ridge National Lab., TN
OSTI Identifier:
7332172
Resource Type:
Journal Article
Journal Name:
J. Biol. Chem.; (United States)
Additional Journal Information:
Journal Volume: 251:15
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ENZYMES; INACTIVATION; THIOLS; ALKYLATION; AMINO ACIDS; BIOCHEMICAL REACTION KINETICS; CARBOXYLIC ACIDS; CHEMICAL REACTIONS; KINETICS; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; REACTION KINETICS; 550200* - Biochemistry

Citation Formats

Hartman, F C, and Norton, I L. Detection of an essential sulfhydryl group in phosphoglycerate mutase with an affinity-labeling reagent. United States: N. p., 1976. Web.
Hartman, F C, & Norton, I L. Detection of an essential sulfhydryl group in phosphoglycerate mutase with an affinity-labeling reagent. United States.
Hartman, F C, and Norton, I L. Thu . "Detection of an essential sulfhydryl group in phosphoglycerate mutase with an affinity-labeling reagent". United States.
@article{osti_7332172,
title = {Detection of an essential sulfhydryl group in phosphoglycerate mutase with an affinity-labeling reagent},
author = {Hartman, F C and Norton, I L},
abstractNote = {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 K/sub m/ 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 /sup 14/C-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 Trp/sub 1/, Lys/sub 1/,Cys(Cm)/sub 1/,Asp/sub 1/,Ser/sub 1/,Glu/sub 2/,Gly/sub 1/,Ala/sub 1/,Leu/sub 1/,Phe/sub 2/.},
doi = {},
journal = {J. Biol. Chem.; (United States)},
number = ,
volume = 251:15,
place = {United States},
year = {1976},
month = {1}
}