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Title: Interaction of isocitrate dehydrogenase with (RS)-3-bromo-2-ketoglutarate. A potential affinity label for. cap alpha. -ketoglutarate binding sites

Abstract

The interaction of oxidize nicotine adenine dinucleotide phosphate dependent isocitrate dehydrogenase (from pig heart) with (RS)-3-bromo-2-ketoglutarate was investigated in an effort to evaluate the reagent's potential as a selective reagent for ..cap alpha..-ketoglutarate binding sites. The enzyme is rapidly inactivated by 0.1 mM bromoketoglutarate at pH 7.4. With increasing concentration of reagent, the reaction shows a rate saturation; the minimum inactivation half-time is 3 min and K/sub inact/ for bromoketoglutarate is 250 ..mu..M. Isocitrate and NADP/sup +/ protect against inactivation, while ketoglutarate does not. When tested in the assay that monitors isocitrate oxidation, bromoketoglutarate is a competitive inhibitor (K/sub i/=100 ..mu..M) of the dehydrogenase. As judged by oxidation of NADPH, bromoketoglutarate is also a substrate for isocitrate dehydrogenase, exhibiting a K/sub m/ of 250 ..mu..M and a V/sub max/ comparable to that for isocitrate oxidation. The reduction of bromoketoglutarate is competitively inhibited by isocitrate (K/sub i/=3 ..mu..M) and ketoglutarate (K/sub i/=50 ..mu..M). Like the enzyme-catalyzed oxidation of isocitrate, the reduction of bromoketoglutarate is stereospecific, requires divalent metal ions, and shows absolute specificity for NADPH. However, since CO/sub 2/ is not required for catalytic turnover of bromoketoglutarate, its reduction is likely comparable to that of oxalosuccinate rather than the reductive carboxylationmore » of ketoglutarate. Although bromoketoglutarate, as a substrate for isocitrate dehydrogenase, clearly has affinity for the active site, the irreversible inactivation of the enzyme by the reagent may result from modification outside the active-site region, since inactivation during catalytic turnover of bromoketoglutarate is not observed.« less

Authors:
Publication Date:
Research Org.:
Oak Ridge National Lab., TN
OSTI Identifier:
6781506
DOE Contract Number:  
W-7405-ENG-26
Resource Type:
Journal Article
Journal Name:
Biochemistry; (United States)
Additional Journal Information:
Journal Volume: 20:4
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; OXIDOREDUCTASES; INTERACTIONS; REAGENTS; BINDING ENERGY; EVALUATION; BIOCHEMICAL REACTION KINETICS; BROMINE COMPOUNDS; GLUTARIC ACID; INHIBITION; KETO ACIDS; CARBOXYLIC ACIDS; DICARBOXYLIC ACIDS; ENERGY; ENZYMES; HALOGEN COMPOUNDS; KINETICS; ORGANIC ACIDS; ORGANIC COMPOUNDS; REACTION KINETICS; 550200* - Biochemistry

Citation Formats

Hartman, F C. Interaction of isocitrate dehydrogenase with (RS)-3-bromo-2-ketoglutarate. A potential affinity label for. cap alpha. -ketoglutarate binding sites. United States: N. p., 1981. Web.
Hartman, F C. Interaction of isocitrate dehydrogenase with (RS)-3-bromo-2-ketoglutarate. A potential affinity label for. cap alpha. -ketoglutarate binding sites. United States.
Hartman, F C. Thu . "Interaction of isocitrate dehydrogenase with (RS)-3-bromo-2-ketoglutarate. A potential affinity label for. cap alpha. -ketoglutarate binding sites". United States.
@article{osti_6781506,
title = {Interaction of isocitrate dehydrogenase with (RS)-3-bromo-2-ketoglutarate. A potential affinity label for. cap alpha. -ketoglutarate binding sites},
author = {Hartman, F C},
abstractNote = {The interaction of oxidize nicotine adenine dinucleotide phosphate dependent isocitrate dehydrogenase (from pig heart) with (RS)-3-bromo-2-ketoglutarate was investigated in an effort to evaluate the reagent's potential as a selective reagent for ..cap alpha..-ketoglutarate binding sites. The enzyme is rapidly inactivated by 0.1 mM bromoketoglutarate at pH 7.4. With increasing concentration of reagent, the reaction shows a rate saturation; the minimum inactivation half-time is 3 min and K/sub inact/ for bromoketoglutarate is 250 ..mu..M. Isocitrate and NADP/sup +/ protect against inactivation, while ketoglutarate does not. When tested in the assay that monitors isocitrate oxidation, bromoketoglutarate is a competitive inhibitor (K/sub i/=100 ..mu..M) of the dehydrogenase. As judged by oxidation of NADPH, bromoketoglutarate is also a substrate for isocitrate dehydrogenase, exhibiting a K/sub m/ of 250 ..mu..M and a V/sub max/ comparable to that for isocitrate oxidation. The reduction of bromoketoglutarate is competitively inhibited by isocitrate (K/sub i/=3 ..mu..M) and ketoglutarate (K/sub i/=50 ..mu..M). Like the enzyme-catalyzed oxidation of isocitrate, the reduction of bromoketoglutarate is stereospecific, requires divalent metal ions, and shows absolute specificity for NADPH. However, since CO/sub 2/ is not required for catalytic turnover of bromoketoglutarate, its reduction is likely comparable to that of oxalosuccinate rather than the reductive carboxylation of ketoglutarate. Although bromoketoglutarate, as a substrate for isocitrate dehydrogenase, clearly has affinity for the active site, the irreversible inactivation of the enzyme by the reagent may result from modification outside the active-site region, since inactivation during catalytic turnover of bromoketoglutarate is not observed.},
doi = {},
journal = {Biochemistry; (United States)},
number = ,
volume = 20:4,
place = {United States},
year = {1981},
month = {1}
}