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 Conservation of Active Sites between the E1 Subunit and Transketolase

Abstract

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 1-lipoyl E2 subunit. While the 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 the lipoyl domain to the H407A E1 variant is much weaker than that to parental E1. At the same time, mass spectrometric measurements clearly demonstrate much impaired reductive acetylation of the independently expressed 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 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.

A Thiamin-bound, Pre-decarboxylation Reaction Intermediate Analogue in the Pyruvate Dehydrogenase E1 Subunit Induces Large Scale Disorder-to-Order Transformations in the Enzyme and Reveals Novel Structural Features in the Covalently Bound Adduct

“…Our previous biochemical studies on the H407A E1 variant clearly indicated its importance in post-decarboxylation steps for the PDHc reaction (19). The mutation was shown to dramatically decrease the rate of reductive acetylation, which could be caused either by direct participation in the catalytic reaction at the active site or by interfering with proper E1-E2 association within the PDHc multienzyme complex.…”

“…We also previously suggested that His-407 functions as a proton donor to a lipoamide sulfur prior to the addition of the enamine to the disulfide (19). It is now tempting to suggest that this protonation occurs after carbon dioxide release because of the availability of His-407 at that instant.…”

“…For crystallization, the protein was dialyzed against 20 mM HEPES buffer at pH 7.0, containing 5 mM dithiothreitol, 0.2% NaN 3 , 0.5 mM EDTA, and 1 M leupeptin. The H407A E1 variant was purified and assayed as reported previously (19). The parental and corresponding variant E1 proteins were independently co-crystallized with PLThDP and Mg 2ϩ by the sitting drop vapor diffusion method under conditions similar to that used for our earlier E1-ThDP crystallographic studies (8).…”

Efficient coupling of catalysis and dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex

“…Although the possibility of electrostatic catalysis in E1ec [facilitated by interaction between H407 and PLThDP ( Fig. 1)] mediated via loop motion has been proposed (3,4,27), the effect of disruption of this interaction is modest [H407A and E401K reduce k cat (E1ec specific) by Ϸ10-fold (4,27)]. Further, it is difficult to model an E1ec reference reaction, because there simply is no reaction in the absence of ThDP, whereas the protein supplies a 10 12 -fold rate acceleration over and above that by ThDP itself (28).…”

Nuclear Magnetic Resonance Evidence for the Role of the Flexible Regions of the E1 Component of the Pyruvate Dehydrogenase Complex from Gram-negative Bacteria

“…We had developed a MALDI-TOF mass spectroscopic method to monitor the reductive acetylation of either the independently expressed lipoyl domain or of the entire E2ec by E1ec and its variants in the presence of pyruvate (18,21). Recently, we extended this method to MALDI-TOF-TOF and Fourier transform mass spectrometry.…”