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. 2008 Jan 29;105(4):1158-63.
doi: 10.1073/pnas.0709328105. Epub 2008 Jan 23.

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

Sachin Kale  1 , Gözde Ulas, Jaeyoung Song, Gary W Brudvig, William Furey, Frank Jordan
Affiliations

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

Sachin Kale et al. Proc Natl Acad Sci U S A. .

Abstract

Protein motions are ubiquitous and are intrinsically coupled to catalysis. Their specific roles, however, remain largely elusive. Dynamic loops at the active center of the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex are essential for several catalytic functions starting from a predecarboxylation event and culminating in transfer of the acetyl moiety to the E2 component. Monitoring the kinetics of E1 and its loop variants at various solution viscosities, we show that the rate of a chemical step is modulated by loop dynamics. A cysteine-free E1 construct was site-specifically labeled on the inner loop (residues 401-413), and the EPR nitroxide label revealed ligand-induced conformational dynamics of the loop and a slow "open <--> close" conformational equilibrium in the unliganded state. An (19)F NMR label placed at the same residue revealed motion on the millisecond-second time scale and suggested a quantitative correlation of E1 catalysis and loop dynamics for the 200,000-Da protein. Thermodynamic studies revealed that these motions may promote covalent addition of substrate to the enzyme-bound thiamin diphosphate by reducing the free energy of activation. Furthermore, the global dynamics of E1 presumably regulate and streamline the catalytic steps of the overall complex by inducing an entirely entropic (nonmechanical) negative cooperativity with respect to substrate binding at higher temperatures. Our results are consistent with, and reinforce the hypothesis of, coupling of catalysis and regulation with enzyme dynamics and suggest the mechanism by which it is achieved in a key branchpoint enzyme in sugar metabolism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Position of the dynamic active-center loops over the active center formed at the interface of the E1ec dimer. The inner (residues 401–413) and outer loops (residues 541–557) are seen ordered over the intermediate analogue PLThDP (yellow-blue-orange sticks), an analogue of the first covalent intermediate LThDP.
Fig. 2.
Fig. 2.
Rate of formation of PLThDP in E1ec and E401K. Fifty-micromolar active sites in 50 mM KH2PO4 (pH 7.0) containing 0.2 mM ThDP and 1.0 mM MgCl2 in one syringe were mixed with 50 μM MAP in the same buffer. (A and B) E1ec at η = 1.0 and 5.3, respectively. (C and D) E401K at η = 1.0 and 5.3, respectively.
Fig. 3.
Fig. 3.
Effect of viscosity on the kcat for E1ec, E401K, and E571A. The solid gray line has a unit slope and represents the diffusion-controlled limit. The nonlinear dashed gray line is a nonlinear fit to the E1ec data, whereas linear dashes gray line is a linear fit to the initial E1ec data points.
Fig. 4.
Fig. 4.
EPR and 19F NMR analysis of conformational dynamics of inner loop in response to MAP and temperature. (A) Spectra of Q408C-MTSL (350 μM) before (i) and after addition (ii) of MAP (1.0 mM) (iii) and (iv) are the simulations (red line) of i and ii, respectively. (B) Spectra of K411C-MTSL (350 μM) before (i) and after addition (ii) of MAP (1.0 mM); iii and iv are simulations (red line) of i and ii, respectively. (C) Effect of MAP addition on the spectra of K411C-TFA. The red line is a spectrum of K411C-TFA and shows three resonances at −8.993, −9.106, and −9.193 ppm. The resonance at −9.193 ppm is due to oxidized free label, did not show any ligand-induced changes, and could be removed by extended washing (D). The black trace is a K411C-TFA (250 μM) after addition of saturating amount (500 μM) of MAP. Spectra were acquired at 30°C and referenced to the external standard trifluoroacetate. The data represent 2,048 transients processed with 5 Hz line broadening and a spectral window of 34,617 Hz. (D) Effect of temperature on the 19F NMR spectra of unliganded K411C-TFA (250 μM). Sample conditions were the same as above.
Fig. 5.
Fig. 5.
Effect of temperature on conformational equilibrium of loop and ligand binding. (A) Temperature-dependent dynamic equilibrium in inner-loop populations of K411C-TFA (250 μM). (Inset) Effect of temperature on the energetics of the population equilibrium. (B) Effect of temperature on the molar heat of binding (ΔHobs) of MAP to E1ec (squares). The solid line represents the linear fit to low-temperature data (5–25°C), whereas the broken line is a fit to higher-temperature data (25–35°C). The transition temperature (Ttran) is a temperature at which step transition (close/open) takes place, and ΔHconf starts to become appreciable and contributes strongly to the observed molar heat of binding (ΔHobs = ΔHbind + ΔHconf). Circles represent the effect of temperature on the molar heat of binding (ΔHobs) of MAP to H407A. A solid line through these points is a linear fit to data.
Fig. 6.
Fig. 6.
Schematic of the effect of temperature on the dynamics of the unliganded and liganded E1ec. In unliganded E1ec (no MAP), the loops exist as a conformational equilibrium of open and closed states (Fig. 4). This equilibrium gradually shifts in favor of the open conformation up to 25°C; there is a step transition in favor of the open conformation at 25°C (Ttran, Fig. 5B). During ligand binding >25°C, the step “close ↔ open” transition gives rise to a configurational enthalpic term (ΔHconf) of a much higher magnitude, resulting from a ligand-induced disorder-to-order transition, which causes progressive reinforcement of observed enthalpy (ΔHobs). However, at 35°C, where the rate of conformational fluctuations of the loop (kex) and hence the coupled rate of covalent substrate addition (k2) increases, the catalytic rate is regulated by temperature induced anticooperative binding of ligand. At this temperature, binding of the second ligand incurs an entropic penalty, which results in 100-fold weaker affinity for the second molecule of ligand giving rise to the observed negative cooperativity.
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