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, [1-13 C]ethanolamine, [2-13 C]ethanolamine, and unlabeled ethanolamine were acquired using RMFQ trapping methods from 10 ms to completion of the reaction. Resolved 13 C hyperfine splitting in EPR spectra of samples prepared with [1-13 C]ethanolamine and the absence of such splitting in spectra of samples prepared with [2-13 C]ethanolamine show that the unpaired electron is localized on C1 (the carbinol carbon) of the substrate. The 13 C splitting from C1 persists from 10 ms throughout the time course of substrate turnover, and there was no evidence for a detectable amount of a product like radical having unpaired spin on C2. These results correct an earlier assignment for this radical intermediate [Warncke 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 1-13 C hyperfine splitting tensor were analyzed through spectral simulations. The sign of the isotropic exchange interaction indicates a weak ferromagnetic coupling of the two unpaired electrons. A Co 2+ to radical distance of 8.7Å was obtained from the magnitude of the dipole-dipole interaction. The orientation of the principal axes of the 13 C 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 z 2 orbital of Co 2+ .Ethanolamine ammonia-lyase (EAL 1 , EC 4.3.1.7) is an AdoCbl dependent enzyme that catalyzes elimination of ammonia from the vicinal position of short chain amino-alcohols such as ethanolamine to give the corresponding oxo products. The functional protein is believed to be a hexamer of αβ-dimers ((αβ) 6 , α ∼50 kDa and β ∼31 kDa) (1,2). EAL is proposed to be an important enzyme in the metabolism of some bacterial species such as Salmonella enterica, which can use ethanolamine, derived from the breakdown of phospholipids, as their sole source † This research was supported by NIH Grant R56-GM35752. ‡ Present address: University of Michigan Medical School, 1150 W. Medical Center Dr., MSRB III, Ann Arbor, MI 48109-0606. * To whom correspondence should be addressed: University of Wisconsin, 1710 University Ave., Madison, WI 53726-4087. Telephone: (608) 262-0509. Fax: (608) 265-2904. E-mail: ghreed@wisc.edu SUPPORTING INFORMATION AVAILABLE 13 C NMR spectra of ( 13 C)ethanolamines and 1 H NMR spectra of ( 13 C)glycine precursors. Stopped flow spectrophotometric assay for Co-carbon bond cleavage in the steady-state with ethanolamine. This material is available free of charge via the ...

Resolution and Characterization of Chemical Steps in Enzyme Catalytic Sequences by Using Low-Temperature and Time-Resolved, Full-Spectrum EPR Spectroscopy in Fluid Cryosolvent and Frozen Solution Systems

“…The Co 2+ -substrate radical pair state, formed by using the two EAL substrates, 2-aminopropanol or aminoethanol, accumulates as the only detectable paramagnetic intermediate during steady-state turnover of EAL at room temperature (Babior et al, 1974; Bandarian & Reed, 2002; Bender et al, 2008). The kinetically unstable substrate radical intermediate state formed from the aminoethanol substrate can be cryotrapped (Warncke et al, 1999), and is stabilized by storage at 77 K. The forward reaction of the substrate radical ensemble can be synchronously initiated at low temperature by a T -step, and the time-evolution of the reaction is monitored by time-resolved, full-spectrum EPR spectroscopy (Zhu & Warncke, 2008, 2010).…”

“…Following Co-C bond cleavage, the low-spin Co 2+ (d 7 ; electron spin, S =1/2) in cobalamin interacts with the radical ( S =1/2) to form a radical pair, which is detected by EPR spectroscopy (Gerfen, 1999). The Co 2+ -substrate radical pair state can be formed and cryotrapped in EAL by using 2-aminopropanol (Babior, Moss, Orme-Johnson, & Beinert, 1974) and aminoethanol (Bender, Poyner, & Reed, 2008; Warncke, Schmidt, & Ke, 1999). Figure 2 shows the EPR spectrum of the Co 2+ -aminopropanol substrate radical pair state.…”

“…Simulations of the radical pair EPR spectra yield the magnetic dipolar interaction coupling constant, D (and, E , in the case of rhombic dipolar interaction symmetry), from which the electron-electron dipolar distances, R ee , and the isotropic exchange coupling constant, J , are obtained (Gerfen, 1999). The cryotrapped Co 2+ -substrate radical pair states in EAL display R ee values of 9–11 Å, and magnitudes of the isotropic exchange interaction, | J |=200–300 MHz (Bender et al, 2008; Canfield & Warncke, 2002; S. C. Ke, 2003).…”

Protein Motions Are Coupled to the Reaction Chemistry in Coenzyme B12‐Dependent Ethanolamine Ammonia Lyase

“…These correspond to chemical changes between cob(II)alamin/substrate radical (SC) pair that accumulates during turnover, [13] and the recombination of the CoÀC bond upon substrate exhaustion [Eq. (2)].…”

“…[5,11] This coupling has been shown to remove magnetic field-sensitivity from the CoÀC homolysis in EAL, by limiting the extent of geminate recombination, and thus favoring the dissociated state. [5,12] EPR spectroscopy has also shown the substrate radical to accumulate during turnover and that it is separated from the Co II by 8.7 , [13] further stabilizing against geminate recombination. Although these factors no doubt contribute to the catalytic power of EAL, there remains little evidence for an explicit role for the protein.…”