Histidine 407, a Phantom Residue in the El Subunit of the Escherichia coli Pyruvate Dehydrogenase Complex, Activates Reductive Acetylation of Lipoamide on the E2 Subunit. An Explanation for Conservation of Active Sites between the El Subunit and...
In: Biochemistry, Jg. 41 (2002-12-31), Heft 52, S. 15459-15467
Online
academicJournal
Zugriff:
Least squares alignment of the E. coli pyruvate dehydrogenase multienzyme complex E1 subunit and yeast transketolase crystal structures indicates a general structural similarity between the two enzymes and provides a plausible location for a short-loop region in the E1 structure that was unobserved due to disorder. The residue H407, located in this region, is shown to be able to penetrate the active site. Suggested by this comparison, the H407A E1 variant was created, and H407 was shown to participate in the reductive acetylation of both an independently expressed lipoyl domain and the intact l-lipoyl E2 subunit. While the H407A substitution only modestly affected the reaction through pyruvate decarboxvlation (ca. 14% activity compared to parental El), the overall complex has a much impaired activity, at most 0.15% compared to parental El. Isothermal titration calorimetry measurements show that the binding of the lipoyl domain to the H407A E1 variant is much weaker than that to parental El. At the same time, mass spectrometric measurements clearly demonstrate much impaired reductive acetylation of the independently expressed lipoyl domain and of the intact 1-1ipoyl E2 by the H407A variant compared to the parental El. A proposal is presented to explain the remarkable conservation of the three-dimensional structure at the active centers of the E. coli E1 subunit and transketolase on the basis of the parallels in the ligation-type reactions carried out and the need to protonate a very weak acid, a dithiolane sulfur atom in the former, and a carbonyl oxygen atom in the latter. [ABSTRACT FROM AUTHOR]
Titel: |
Histidine 407, a Phantom Residue in the El Subunit of the Escherichia coli Pyruvate Dehydrogenase Complex, Activates Reductive Acetylation of Lipoamide on the E2 Subunit. An Explanation for Conservation of Active Sites between the El Subunit and...
|
---|---|
Autor/in / Beteiligte Person: | Nemeria, Natalia ; Arjunan, Palaniappa ; Brunskill, Andrew ; Sheibani, Farzad ; Wei, Wen ; Yan, Yan ; Zhang, Sheng ; Jordan, Frank ; Furey, William |
Link: | |
Zeitschrift: | Biochemistry, Jg. 41 (2002-12-31), Heft 52, S. 15459-15467 |
Veröffentlichung: | 2002 |
Medientyp: | academicJournal |
ISSN: | 0006-2960 (print) |
DOI: | 10.1021/bi0205909 |
Schlagwort: |
|
Sonstiges: |
|