Histidine 407, a Phantom Residue in the E1 Subunit of the Escherichia coli Pyruvate Dehydrogenase Complex, Activates Reductive Acetylation of Lipoamide on the E2 Subunit. An Explanation for Con
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- 作者：Natalia Nemeria ; Palaniappa Arjunan ; Andrew Brunskill ; Farzad Sheibani ; Wen Wei ; Yan Yan ; Sheng Zhang ; Frank Jordan ; and William Furey
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：December 31, 2002
- 年：2002
- 卷：41
- 期：52
- 页码：15459 - 15467
- 全文大小：325K
- 年卷期：v.41,no.52(December 31, 2002)
- ISSN：1520-4995
文摘
Least squares alignment of the E. coli pyruvate dehydrogenase multienzyme complex E1 subunitand yeast transketolase crystal structures indicates a general structural similarity between the two enzymesand provides a plausible location for a short-loop region in the E1 structure that was unobserved due todisorder. The residue H407, located in this region, is shown to be able to penetrate the active site. Suggestedby this comparison, the H407A E1 variant was created, and H407 was shown to participate in the reductiveacetylation of both an independently expressed lipoyl domain and the intact 1-lipoyl E2 subunit. Whilethe H407A substitution only modestly affected the reaction through pyruvate decarboxylation (ca. 14%activity compared to parental E1), the overall complex has a much impaired activity, at most 0.15%compared to parental E1. Isothermal titration calorimetry measurements show that the binding of thelipoyl domain to the H407A E1 variant is much weaker than that to parental E1. At the same time, massspectrometric measurements clearly demonstrate much impaired reductive acetylation of the independentlyexpressed lipoyl domain and of the intact 1-lipoyl E2 by the H407A variant compared to the parental E1.A proposal is presented to explain the remarkable conservation of the three-dimensional structure at theactive centers of the E. coli E1 subunit and transketolase on the basis of the parallels in the ligation-typereactions 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.