Effect of substitutions in the thiamin diphosphate-magnesium fold on the activation of the pyruvate dehydrogenase complex from Escherichia coli by cofactors and substrate.

The Journal of Biological Chemistry
J YiJ R Guest

Abstract

The homotropic regulation of the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) by its coenzyme thiamin diphosphate and its substrate pyruvate was re-examined with complexes containing three and one lipoyl domains per E2 chain, and several variants of the latter, containing substitutions in the putative thiamin diphosphate fold of E1 (G231A, G231S, C259S, C259N, and N258Q). It was found that all of the E1 variants had significantly reduced specific activities, as reported elsewhere (Russell, G. C., Machado, R. S., and Guest, J. R. (1992) Biochem. J. 287, 611-619). In addition, extensive kinetic studies were performed in an attempt to determine the effects of the amino acid substitutions on the Hill coefficients with respect to thiamin diphosphate and pyruvate. All but one of the variants were incapable of being saturated with thiamin diphosphate, even at concentrations > 5 mM. Most importantly, the striking activation lag phase lasting for many seconds in the parental complexes containing three and one lipoyl domains per E2 chain was totally eliminated in the variants. Furthermore, activation by the coenzyme was localized to the E1 subunit, because resolved E1 exhibits virtually the same behavior during the ...Continue Reading

References

Sep 11, 1989·FEBS Letters·C F HawkinsR N Perham
Apr 1, 1969·European Journal of Biochemistry·J Bremer
May 10, 1968·Biochemical and Biophysical Research Communications·E R SchwartzL J Reed
Dec 9, 1970·Biochemical and Biophysical Research Communications·G A Kochetov, R A Usmanov
Oct 3, 1983·European Journal of Biochemistry·P E StephensJ R Guest
Jul 31, 1980·Biochemical and Biophysical Research Communications·H Bisswanger
Feb 1, 1995·Protein Expression and Purification·L G KorotchkinaM S Patel
Oct 15, 1971·FEBS Letters·G H. Gibbon, S J. Pirt

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Citations

Jun 27, 2007·Biotechnology and Bioengineering·Christophe ChassagnoleMatthias Reuss
May 23, 2001·Archives of Biochemistry and Biophysics·S LiuT E Roche
Aug 16, 2005·Biochemistry. Biokhimii︠a︡·O A EsakovaG A Kochetov

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