S-2-bromo-acyl-CoA analogues are affinity labels for the medium-chain acyl-CoA dehydrogenase from pig kidney.
S-2-Br-hexanoyl-CoA and the branched chain isomer S-2-Br-4-methyl-pentanoyl-CoA are affinity labels of the medium-chain acyl-CoA dehydrogenase from pig kidney. The straight chain thioester is both a substrate and an irreversible inhibitor of the dehydrogenase. Inactivation of the enzyme is biphasic and is half-complete in 4 min at pH 6.5, 25 degrees C. Although S-2-Br-hexanoyl-CoA can partially reduce the FAD prosthetic group of the dehydrogenase, inactivation results from attachment of one molecular of inhibitor per subunit of the oxidized enzyme. The branched chain analogue is a very weak substrate of the dehydrogenase (0.1% that of octanoyl-CoA), but is almost as effective an inhibitor of the dehydrogenase. Incubation experiments with [14C]S-2-Br-methyl-pentanoyl-CoA followed by the isolation of radiolabeled peptide show that modification of the active site base, GLU376, is responsible for enzyme inactivation. The data are compatible with a simple nucleophilic attack of the carboxylate base on the C-2 atom of these 2-Br-analogues.
Haeffner-Gormley
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L
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Cummings
J G
JG
Thorpe
C
C
D000214:Acyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology*; D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D013053:Spectrophotometry; D013552:Swine; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D000214:Acyl Coenzyme A; D000345:Affinity Labels; D010446:Peptide Fragments; C092540:S-2-bromo-4-methylpentanoyl-coenzyme A; C092539:S-2-bromohexanoyl-coenzyme A; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase; D014357:Trypsin