Multiple roles of mobile active center loops in the E1 component of the Escherichia coli pyruvate dehydrogenase complex - Linkage of protein dynamics to catalysis

Abstract

The region encompassing residues 401-413 on the E1 component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli comprises a loop (the inner loop) which was not seen in the X-ray structure in the presence of thiamin diphosphate, the required cofactor for the enzyme. This loop is seen in the presence of a stable analogue of the pre-decarboxylation intermediate, the covalent adduct between the substrate analogue methyl acetylphosphonate and thiamin diphosphate, C2α-phosphonolactylthiamin diphosphate. It has been shown that the residue H407 and several other residues on this loop are required to reduce the mobility of the loop so electron density corresponding to it can be seen once the pre-decarboxylation intermediate is formed. Concomitantly, the loop encompassing residues 541-557 (the outer loop) appears to work in tandem with the inner loop and there is a hydrogen bond between the two loops ensuring their correlated motion. The inner loop was shown to: a) sequester the active center from carboligase side reactions; b) assist the interaction between the E1 and the E2 components, thereby affecting the overall reaction rate of the entire multienzyme complex; c) control substrate access to the active center. Using viscosity effects on kinetics it was shown that formation of the pre-decarboxylation intermediate is specifically affected by loop movement. A cysteine-less variant was created for the E1 component, onto which cysteines were substituted at selected loop positions. Introducing an electron spin resonance spin label and an (19)F NMR label onto these engineered cysteines, the loop mobility was examined: a) both methods suggested that in the absence of ligand, the loop exists in two conformations; b) line-shape analysis of the NMR signal at different temperatures, enabled estimation of the rate constant for loop movement, and this rate constant was found to be of the same order of magnitude as the turnover number for the enzyme under the same conditions. Furthermore, this analysis gave important insights into rate-limiting thermal loop dynamics. Overall, the results suggest that the dynamic properties correlate with catalytic events on the E1 component of the pyruvate dehydrogenase complex.

Figure 1

Figure 1a. Stereo illustration of an E. coli PDHc E1 subunit superimposed on an S. cerevisiae TK subunit. Colors are green and black for the E1 and TK structures, respectively. The ThDP cofactors are shown in blue. Figure 1b. Stereo illustration of the active site environment in E. coli PDHc E1 with the TK structure superimposed. E1 residues numbered less than 471 are from the N-terminal of one subunit, and those greater than 470 are from the middle domain of the “other” subunit forming the active dimer. The main chain and histidine residue unobserved in E1 but present in TK are shown in magenta. The cofactor ThDP is shown in green.

Figure 1

Figure 1a. Stereo illustration of an E. coli PDHc E1 subunit superimposed on an S. cerevisiae TK subunit. Colors are green and black for the E1 and TK structures, respectively. The ThDP cofactors are shown in blue. Figure 1b. Stereo illustration of the active site environment in E. coli PDHc E1 with the TK structure superimposed. E1 residues numbered less than 471 are from the N-terminal of one subunit, and those greater than 470 are from the middle domain of the “other” subunit forming the active dimer. The main chain and histidine residue unobserved in E1 but present in TK are shown in magenta. The cofactor ThDP is shown in green.

Figure 2

2Fo-Fc omit electron density map contoured at 1σ for part of the region that became ordered in the presence of PLThDP. The reaction intermediate analogue PLThDP is shown as a ball and stick figure. Note the hydrogen bond between the analogue and His407.

Figure 3

Location of the inner loop on E1ec. A] Stereo view of inner loop and intermediate (1′–4′ imino tautomer of PLThDP) hydrogen bonding in active site loops 401–413 (yellow) from subunit A (green) and 541–557 (blue) from subunit B (red) of E1ec. PLThDP (analogue of LThDP) is seen in the active site. Hydrogen bonding (…) between loops and between H407 and PLThDP can be seen. B] Surface view of the E1ec showing active site channel and the positions of two loops [401–413 in blue from subunit α_2a (white) and 541–557 in red from subunit α_2b (green)]. The interaction of two loops creates a new surface leading to active site formed at the interface of two subunits [7]. The positively charged residues K410 and K411 lining the active site cavity can be seen in yellow. Intermediate analogue, in presence of which loops gets organized, can be seen deep inside active site cavity. Coordinates of PDB file 2G25 was used to create figure with the help of Pymol.

Figure 4

Stabilization of transient Michaelis complex with pyruvate in low activity loop variants. Representative trace of each variant at a particular active site concentration is shown. In actual titration intensity of negative peak (centered at 327 nm) was proportional to active site concentration for all the variants in the figure.

Figure 5

5A. Lineshape simulations for unliganded K411C labeled with TFA. The smooth line is a simulated spectrum. Data was simulated using WinDNMR-Pro assuming two state ‘open close’ transitions. While linewidth and resonance frequency were kept constant the relative population of each state, baseline intensity and the exchange rates between states were allowed to vary. 5B. Effect of temperature on the k_cat of K411C-TFA. The data are means ± SE from at least 3 measurements.

Scheme I

Mechanism of bacterial pyruvate dehydrogenase complex with role of thiamin diphosphate

Scheme II

Mechanism of formation of 1′,4′-iminolactylThDP and 1′,4′-iminophosphonolactylThDP as a result of covalent addition of substrate pyruvate and substrate analogue methyl acetylphosphonate (MAP), respectively to enzyme bound ThDP.