PMID: 6161006Nov 1, 1980Paper

Fluorescence polarization and energy-transfer studies on the pyruvate dehydrogenase complex of Escherichia coli

European Journal of Biochemistry
W H ScoutenC Veeger

Abstract

We have attached eosin maleimide specifically to the lipoyl group of the pyruvate dehydrogenase complex isolated from Escherichia coli. Using this as the fluorescence acceptor and the intrinsic FAD of the lipoamide dehydrogenase subunit as the fluorescence donor, we confirmed previous measurements with other probes, in which it was suggested that the flavin moiety is at a substantial distance (over 4.5 nm) from the labeled lipoyl group. Since the lipoyl group must apply electrons to the FAD during the catalytic decarboxylation of pyruvate, we have investigated several potential mechanisms whereby this could happen. Movement within the complex, possibly triggered by the presence of substrate, seemed to be a strong possibility. Complex labeled with fluorophores on the accessible sulfhydryls, or on the lipoyl functions, did not give evidence of such triggering upon addition of substrate as judged by both static and dynamic fluorescence depolarization. The mobility of the subunits of labeled lipoamide dehydrogenase exceeded that expected for the total complex. Pyrene maleimide bound to the lipoyl functions also exhibited considerably faster rotations than the predicted one of the whole complex (tau c > 3 micros). This suggests that...Continue Reading

References

Jan 1, 1979·Proceedings of the National Academy of Sciences of the United States of America·I MunroL Stryer
Oct 1, 1978·Proceedings of the National Academy of Sciences of the United States of America·K J Angelides, G G Hammes
Oct 9, 1973·Biochemistry·E N Hudson, G Weber
Jun 1, 1974·Analytical Biochemistry·A Grinvald, I Z Steinberg
Jan 1, 1980·Methods in Enzymology·A J Visser, F Müller

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Citations

May 1, 1991·Biotechnology and Bioengineering·D L Meadows, J S Schultz
Dec 1, 1981·European Journal of Biochemistry·A J VisserD Lavalette
Jan 1, 1982·Annals of the New York Academy of Sciences·H J BosmaG Voordouw
Oct 21, 2006·Biochemical Society Transactions·M Smolle, J G Lindsay
Jul 10, 1998·Biochimica Et Biophysica Acta·A de KokA H Westphal

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