Identification of the Substrate Radical Intermediate Derived from Ethanolamine during Catalysis by Ethanolamine Ammonia-Lyase

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• 作者：Gü ; neş ; Bender ; Russell R. Poyner ; George H. Reed
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：October 28, 2008
• 年：2008
• 卷：47
• 期：43
• 页码：11360-11366
• 全文大小：177K
• 年卷期：v.47,no.43(October 28, 2008)
• ISSN：1520-4995

文摘

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 Å 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 ∼98° with the unique axis of the d_z² orbital of Co²⁺.