Interaction between catalytic and regulatory sites of the pyruvate dehydrogenase from Escherichia coli studied by the ESR technique.
The accessibility of sulfhydryl groups at the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was reinvestigated. Hydrophobic interactions appear to control the reactivity of an essential cysteine residue at the active site with thiol reagents. This explains why the essential cysteine residue reacts only with thiol reagents of minor polarity, like p-hydroxymercuribenzoate or phenylmercuric nitrate, but not with Ellman's reagent or jodoacetamide. The pyruvate dehydrogenase component was modified with a nitroxide derivative of p-hydroxymercuribenzoate. The ESR spectrum of the spin-labelled enzyme changed dramatically upon addition of the cofactors thiamine diphosphate and Mg2+. Obviously spin-spin interaction occurs under these conditions caused by a transition of an inactive to an active state of the enzyme. The same conformational change is observed when the allosteric activator AMP instead of the cofactors was bound to the enzyme. The implications of these results for the allosteric regulation of the pyruvate dehydrogenase complex are discussed.
Graupe
K
K
Physiologisch-chemisches Institut, Universität Tübingen, F.R.G.
Trommer
W E
WE
Bisswanger
H
H
D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007202:Indicators and Reagents; D007460:Iodoacetamide / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D013113:Spin Labels
D006902:Hydroxymercuribenzoates; D007202:Indicators and Reagents; D007461:Iodoacetates; D011768:Pyruvate Dehydrogenase Complex; D013113:Spin Labels; C030601:4-hydroxymercuribenzoate; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide; D019807:Iodoacetic Acid; D007460:Iodoacetamide