PMID: 9428672Jan 15, 1998Paper

Expression and characterisation of the homodimeric E1 component of the Azotobacter vinelandii pyruvate dehydrogenase complex

European Journal of Biochemistry
Annechien F HengeveldA de Kok

Abstract

We have cloned and sequenced the gene encoding the homodimeric pyruvate dehydrogenase component (E1p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and expressed and purified the E1p component in Escherichia coli. Cloned E1p can be used to fully reconstitute complex activity. The enzyme was stable in high ionic strength buffers, but was irreversibly inactivated when incubated at high pH, which presumably was caused by its inability to redimerize correctly. This explains the previously found low stability of the wild-type E1p component after resolution from the complex at high pH. Cloned E1p showed a kinetic behaviour exactly like the wild-type complex-bound enzyme with respect to its substrate (pyruvate), its allosteric properties, and its effectors. These experiments show that acetyl coenzyme A acts as a feedback inhibitor by binding to the E1p component. Limited proteolysis experiments showed that the N-terminal region of E1p was easily removed. The resulting protein fragment was still active with artificial electron acceptors but had lost its ability to bind to the core component (E2p) and thus reconstitute complex activity. E1p was protected against proteolysis by E2p. The allosteric effector pyruvate ch...Continue Reading

References

Nov 15, 1975·European Journal of Biochemistry·T W BrestersC Veeger
Sep 11, 1989·FEBS Letters·C F HawkinsR N Perham
Dec 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·M A Honma, F M Ausubel
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Jan 1, 1953·Scandinavian Journal of Clinical and Laboratory Investigation·J GOA

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Citations

Oct 27, 1999·Proceedings of the National Academy of Sciences of the United States of America·K RegnströmB K Burgess
May 5, 2000·Molecular Plant-microbe Interactions : MPMI·D CabanesJ Batut
Mar 19, 2015·Journal of Proteomics·Katalin VölgyiArpád Dobolyi
Oct 16, 1999·European Journal of Biochemistry·A F HengeveldA de Kok
Jul 10, 1998·Biochimica Et Biophysica Acta·A de KokA H Westphal

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