Open AccessMeasurements of electron spin resonance with the pyruvate dehydrogenase complex from Escherichia coli
Author(s) -
SCHRENK Dieter F.,
BISSWANGER Hans
Publication year - 1984
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1984.tb08406.x
Subject(s) - dihydrolipoyl transacetylase , pyruvate dehydrogenase complex , pyruvate dehydrogenase phosphatase , chemistry , pyruvate decarboxylation , binding site , dehydrogenase , branched chain alpha keto acid dehydrogenase complex , allosteric regulation , pyruvate dehydrogenase kinase , affinity label , biochemistry , oxoglutarate dehydrogenase complex , stereochemistry , enzyme
Binding of the feedback inhibitor acetyl‐coenzyme A to the pyruvate dehydrogenase complex from Escherichia coli was studied by electron spin resonance spectroscopy with the spin‐labelled acetyl‐CoA analogue 3‐carboxy‐2,2,5,5‐tetramethylpyrrolidine‐1‐oxyl‐CoA‐thioester. The spin‐labelled compound binds to the pyruvate dehydrogenase component of the enzyme complex and this binding can be reversed by acetyl‐CoA, while CoA has no effect. AMP and fructose 1, 6‐bisphosphate, which are both activators of the pyruvate dehydrogenase complex, exhibit a partial competition with the spin‐labelled acetyl‐CoA analogue and it could be shown that both activators act essentially by reversion of the feedback inhibition of acetyl‐CoA. The binding site for these activators seems to overlap with the acetyl‐CoA binding site, possibly by a common phosphate attachment point. No competition for binding to the feedback inhibition site exists with pyruvate, thiamine diphosphate, magnesium ions and with the fluorescent chromophore 8‐anilino‐1‐naphthalene sulfonic acid. Thus, the feedback inhibition site proves to be a true allosteric regulatory site, which appears to be completely separate from the catalytic site on the pyruvate dehydrogenase component. The spin‐labelled acetyl‐CoA analogue binds also to the product binding site of acetyl‐CoA on the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex. Two binding sites per polypeptide chain with identical affinities on this enzyme component were found and the binding of the analogue can be inhibited by acetyl‐CoA as well as by CoA.