Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.1.1.41 (isocitrate dehydrogenase)
 3,101 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pig heart NAD-dependent isocitrate dehydrogenase is inactivated by adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate] (AMPS-BDB) with incorporation of 1.78 mol of reagent/mol of average subunit. Complete protection against the inactivation is provided by 20 mM isocitrate + 1 mM Mn2+, and the incorporation is decreased to about 1.3 mol of reagent/mol of average subunit. The addition of NAD, NADH, or Mn2+ alone has little effect on the functional changes produced by AMPS-BDB, while ADP gives only partial protection against the inactivation. The ability of ADP to decrease the Km for isocitrate is not affected by the AMPS-BDB modification of the enzyme. These results indicate that the isocitrate substrate site is the target of AMPS-BDB. The enzyme has three types of subunits with a tetramer having the composition alpha2 beta gamma. Here, [2-3H]AMPS-BDB-modified subunits are separated by HPLC on a C4 reverse-phase column, after the treatment of the modified enzyme with 4 M urea. The predominant radioactivity is distributed in alpha and gamma subunits. However, evidence based on recombination of subunits from modified and unmodified enzymes indicates that only labeling of the alpha subunit is responsible for inactivation by AMPS-BDB. Subsequently, the separated modified subunits were chemically cleaved by CNBr and then purified by HPLC using a C18 column. The labeled peptides were further digested by pepsin, purified by HPLC, and sequenced. These results indicate that R88 and R98 from the alpha subunit are the major targets of AMPS-BDB which cause inactivation and that these are at or near the isocitrate site of the enzyme.
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PMID:Identification of the subunit and important target peptides of pig heart NAD-dependent isocitrate dehydrogenase modified by the affinity label adenosine 5'-O-[S-(4-bromo-2, 3-dioxobutyl)thiophosphate]. 957 72

A new photoaffinity label, adenosine 2'-monophosphate, 5'-O-[S-(4-succinimidyl-benzophenone)thiophosphate] (2'-P-AMPS-Succ-BP), has been synthesized by an initial thiophosphorylation of 2'-AMP with PSCl(3) to form 2'-AMP-5'-thiophosphate (2'-AMP-5'-SP), followed by a coupling reaction of 2'-AMP-5'-SP with benzophenone-4-maleimide to produce 2'-P-AMPS-Succ-BP. This product and its precursor were characterized by thin-layer chromatography, (31)P NMR, phosphorus analysis, and electron-spray mass spectroscopy. 2'-P-AMPS-Succ-BP functions as a photoaffinity label of porcine NADP-specific isocitrate dehydrogenase. To obtain reaction with other amino acids, Cys269 and Cys379, the most reactive cysteines of this enzyme, were mutated to yield a double mutant enzyme (C269A/C379S) exhibiting comparable activity and kinetic parameters to those of wild-type enzyme. 2'-P-AMPS-Succ-BP inactivates C269A/C379S enzyme upon UV irradiation. The reaction exhibits a nonlinear relationship of k(inact) versus [2'-P-AMPS-Succ-BP] with K(R) = 12 microM and k(max) = 0.0275 min(-1). NADP, NADPH, or 2'-monophospho-adenosine 5'-diphosphoribose protects the enzyme against 2'-P-AMPS-Succ-BP inactivation. The ligand protection studies suggest that 2'-P-AMPS-Succ-BP binds to the porcine enzyme at the site best occupied by NADP/NADPH. The dimeric C269A/C379S isocitrate dehydrogenase incorporates 1.0 mol of 2'-P-[(35)S]AMPS-Succ-BP/mol enzyme dimer concomitant with complete loss of enzyme activity. The new photoaffinity label may be generally useful to identify important amino acid residues of NADP-specific enzymes.
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PMID:Adenosine 2'-monophosphate, 5'-O-[S-(4-succinimidylbenzophenone)-thiophosphate]: a new photoaffinity label for the coenzyme site of porcine NADP-specific isocitrate dehydrogenase. 1589 34