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Identification of the substrate radical intermediate derived from ethanolamine during catalysis by ethanolamine ammonia-lyase.

ABSTRACT: Rapid-mix freeze-quench (RMFQ) methods and electron paramagnetic resonance (EPR) spectroscopy have been used to characterize the steady-state radical in the deamination of ethanolamine catalyzed by adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL). EPR spectra of the radical intermediates formed with the substrates, [1-13C]ethanolamine, [2-13C]ethanolamine, and unlabeled ethanolamine were acquired using RMFQ trapping methods from 10 ms to completion of the reaction. Resolved 13C hyperfine splitting in EPR spectra of samples prepared with [1-13C]ethanolamine and the absence of such splitting in spectra of samples prepared with [2-13C]ethanolamine show that the unpaired electron is localized on C1 (the carbinol carbon) of the substrate. The 13C splitting from C1 persists from 10 ms throughout the time course of substrate turnover, and there was no evidence of a detectable amount of a product like radical having unpaired spin on C2. These results correct an earlier assignment for this radical intermediate [Warncke, K., et al. (1999) J. Am. Chem. Soc. 121, 10522-10528]. The EPR signals of the substrate radical intermediate are altered by electron spin coupling to the other paramagnetic species, cob(II)alamin, in the active site. The dipole-dipole and exchange interactions as well as the 1-13C hyperfine splitting tensor were analyzed via spectral simulations. The sign of the isotropic exchange interaction indicates a weak ferromagnetic coupling of the two unpaired electrons. A Co2+-radical distance of 8.7 A was obtained from the magnitude of the dipole-dipole interaction. The orientation of the principal axes of the 13C hyperfine splitting tensor shows that the long axis of the spin-bearing p orbital on C1 of the substrate radical makes an angle of approximately 98 degrees with the unique axis of the d(z2) orbital of Co2+.

SUBMITTER: Bender G

PROVIDER: S-EPMC2631207 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Identification of the substrate radical intermediate derived from ethanolamine during catalysis by ethanolamine ammonia-lyase.

Bender Güneş G Poyner Russell R RR Reed George H GH

Biochemistry 20081001 43

Rapid-mix freeze-quench (RMFQ) methods and electron paramagnetic resonance (EPR) spectroscopy have been used to characterize the steady-state radical in the deamination of ethanolamine catalyzed by adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL). EPR spectra of the radical intermediates formed with the substrates, [1-13C]ethanolamine, [2-13C]ethanolamine, and unlabeled ethanolamine were acquired using RMFQ trapping methods from 10 ms to completion of the reaction. Resolved ...[more]

PMID: 18826329

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Project description:The adenosylcobalamin- (coenzyme B12) dependent ethanolamine ammonia-lyase (EAL) plays a key role in aminoethanol metabolism, associated with microbiome homeostasis and Salmonella- and Escherichia coli-induced disease conditions in the human gut. To gain molecular insight into these processes toward development of potential therapeutic targets, reactions of the cryotrapped (S)-2-aminopropanol substrate radical EAL from Salmonella typhimurium are addressed over a temperature (T) range of 220-250 K by using T-step reaction initiation and time-resolved, full-spectrum electron paramagnetic resonance spectroscopy. The observed substrate radical reaction kinetics are characterized by two pairs of biexponential processes: native decay to diamagnetic products and growth of a non-native radical species and Co(II) in cobalamin. The multicomponent low-T kinetics are simulated by using a minimal model, in which the substrate-radical macrostate, S?, is partitioned by a free-energy barrier into two sequential microstates: 1) S1?, a relatively high-entropy/high-enthalpy microstate with a protein configuration that captures the nascent substrate radical in the terminal step of radical-pair separation; and 2) S2?, a relatively low-enthalpy/low-entropy microstate with a protein configuration that enables the rearrangement reaction. The non-native, destructive reaction of S1? at T ? 250 K is caused by a prolonged lifetime in the substrate-radical capture state. Monotonic S? decay over 278-300 K indicates that the free-energy barrier to S1? and S2? interconversion is latent at physiological T-values. Overall, the low-temperature studies reveal two protein-configuration microstates and connecting protein-configurational transitions that specialize the S? macrostate for the dual functional roles of radical capture and rearrangement enabling. The identification of new, to our knowledge, intermediate states and specific protein-fluctuation contributions to the reaction coordinate represent an advance toward development of novel therapeutic targets in EAL.

Project description:The decay kinetics of the aminoethanol-generated Co(II)-substrate radical pair catalytic intermediate in ethanolamine ammonia-lyase from Salmonella typhimurium have been measured on timescales of <10(5) s in frozen aqueous solution from 190 to 217 K. X-band continuous-wave electron paramagnetic resonance (EPR) spectroscopy of the disordered samples has been used to continuously monitor the full radical pair EPR spectrum during progress of the decay after temperature step reaction initiation. The decay to a diamagnetic state is complete and no paramagnetic intermediate states are detected. The decay exhibits three kinetic regimes in the measured temperature range, as follows. i), Low temperature range, 190 < or = T < or = 207 K: the decay is biexponential with constant fast (0.57 +/- 0.04) and slow (0.43 +/- 0.04) phase amplitudes. ii), Transition temperature range, 207 < T < 214 K: the amplitude of the slow phase decreases to zero with a compensatory rise in the fast phase amplitude, with increasing temperature. iii), High temperature range, T > or = 214 K: the decay is monoexponential. The observed first-order rate constants for the monoexponential (k(obs,m)) and the fast phase of the biexponential decay (k(obs,f)) adhere to the same linear relation on an lnk versus T(-1) (Arrhenius) plot. Thus, k(obs,m) and k(obs,f) correspond to the same apparent Arrhenius prefactor and activation energy (logA(app,f) (s(-1)) = 13.0, E(a,app,f) = 15.0 kcal/mol), and therefore, a common decay mechanism. We propose that k(obs,m) and k(obs,f) represent the native, forward reaction of the substrate through the radical rearrangement step. The slow phase rate constant (k(obs,s)) for 190 < or = T < or = 207 K obeys a different linear Arrhenius relation (logA(app,s) (s(-1)) = 13.9, E(a,app,s) = 16.6 kcal/mol). In the transition temperature range, k(obs,s) displays a super-Arrhenius increase with increasing temperature. The change in E(a,app,s) with temperature and the narrow range over which it occurs suggest an origin in a liquid/glass or dynamical transition. A discontinuity in the activation barrier for the chemical reaction is not expected in the transition temperature range. Therefore, the transition arises from a change in the properties of the protein. We propose that a protein dynamical contribution to the reaction, which is present above the transition temperature, is lost below the transition temperature, owing to an increase in the activation energy barrier for protein motions that are coupled to the reaction. For both the fast and slow phases of the low temperature decay, the dynamical transition in protein motions that are obligatorily coupled to the reaction of the Co(II)-substrate radical pair lies below 190 K.

Dataset Information

Identification of the substrate radical intermediate derived from ethanolamine during catalysis by ethanolamine ammonia-lyase.

Publications

Identification of the substrate radical intermediate derived from ethanolamine during catalysis by ethanolamine ammonia-lyase.

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