A Dynamic Loop at the Active Center of the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Modulates Substrate Utilization and Chemical Communication with the E2 Component

Abstract

Our crystallographic studies have shown that two active center loops (an inner loop formed by residues 401-413 and outer loop formed by residues 541-557) of the E1 component of the Escherichia coli pyruvate dehydrogenase complex become organized only on binding a substrate analog that is capable of forming a stable thiamin diphosphate-bound covalent intermediate. We showed that residue His-407 on the inner loop has a key role in the mechanism, especially in the reductive acetylation of the E. coli dihydrolipoamide transacetylase component, whereas crystallographic results showed a role of this residue in a disorder-order transformation of these two loops, and the ordered conformation gives rise to numerous new contacts between the inner loop and the active center. We present mapping of the conserved residues on the inner loop. Kinetic, spectroscopic, and crystallographic studies on some inner loop variants led us to conclude that charged residues flanking His-407 are important for stabilization/ordering of the inner loop thereby facilitating completion of the active site. The results further suggest that a disorder to order transition of the dynamic inner loop is essential for substrate entry to the active site, for sequestering active site chemistry from undesirable side reactions, as well as for communication between the E1 and E2 components of the E. coli pyruvate dehydrogenase multienzyme complex.The Escherichia coli pyruvate dehydrogenase multienzyme complex is comprised of the following three enzyme components: E1ec 2 (24 copies, 99,474 Da), E2ec (24 copies, 65,959 Da), and E3ec (12 copies, 50,554 Da) (1-3). The complex catalyzes the oxidative decarboxylation of pyruvate according to Equation 1 (4),The components catalyze parts of the overall reaction (5-7) as shown in Equations 2-6,The E1ec catalyzes thiamin diphosphate (ThDP)-dependent decarboxylation of pyruvate whose product reductively acetylates the lipoamide group of E2ec (supplemental Scheme I). The crystal structure of E1ec with ThDP bound revealed the absence of defined electron density for regions encompassing residues 1-55, 401-413, and 541-557 (8). The first of these regions, the N-terminal one, was shown to interact with E2ec (9). A BLAST search and structure alignment of E1ec with transketolase suggested that a highly conserved residue His-407 of the 401-413 disordered loop (henceforth the "inner" loop) * This work was supported by National Institutes of Health Grants GM 50380and GM 61791. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

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

“…5B and ref. 4) and crystal structure (3,4)], the K d PLThDP significantly increased, whereas the rate of PLThDP formation (in E401K) significantly decreased. This suggests that preequilibrium may ''be harvested for catalytic turnover'' (25).…”

“…We have shown that on E1ec, it is difficult to observe the AP form. However, addition of pyruvate to the E401K loop variant under a variety of conditions produced the negative CD band corresponding to MC, stabilized as a result of very slow catalysis caused by impaired loop dynamics (4). It could be shown that the MC is fully formed in E1ec and E401K, as measured with SF-CD, within the dead time of the instrument (1 ms) (SI Fig.…”

“…In the crystal structures of E1ec loop variants studied to date, both loops are seen disordered even when the only stabilizing interactions that are disrupted by the substitutions are between the inner loop and the protein (E401K and K403E) (4) or between the inner loop and intermediate analogue (H407A) (3) rather than between two loops. Therefore, the dynamics of the two active-center loops appear to be concerted, they work in tandem.…”

“…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).…”

“…This ordered conformation could be associated with a closed one, because it protects the active site from solvent, in contrast to a disordered (or open) conformation, in which the enamine reacts with excess pyruvate in the ''carboligation'' side reaction (4). Also, because the fraction of immobile component increased with concomitant decrease in the fraction of mobile component (their respective R values showing reciprocal changes), we hypothesize that these components might represent two environments encountered by the probe reflecting different conformations.…”