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

Ethanolamine ammonia-lyase (EAL1, 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 of carbon, nitrogen, and energy (3). Catalysis by EAL with various substrates and inactivation of EAL with substrate analogues have been studied extensively by kinetic methods, and radical states occurring during catalysis have been probed by EPR spectroscopy (4-9). The putative catalytic cycle of EAL starts with the formation of a highly reactive 5′-deoxyadenosyl radical and cob(II)alamin after homolysis of the Co-carbon bond of the cofactor (Scheme 1) (10). The transient 5′-deoxyadenosyl radical abstracts the pro-S hydrogen atom from C1 of the substrate to create the initial substrate radical (11). Abstraction of the hydrogen atom from C1 is kinetically coupled to the Co-carbon bond homolysis and pulls the Co-carbon bond cleavage process forward (6). The substrate radical rearranges to a product-like cabinolamine radical, which abstracts a hydrogen atom from the 5′ carbon of 5′-deoxyadenosine to give a carbinolamine. Breakdown of the carbinolamine to ammonia and acetaldehyde and recombination of the 5′-deoxyadenosyl radical with cob(II)alamin complete the catalytic cycle. Details of the radical rearrangement step are not yet clear. The product radical was proposed to be a carbinolamine radical on the basis of theoretical energy calculations of possible intermediates and transition states (12-14). Scheme 1 EPR spectroscopy reveals the presence of organic radical intermediates spin coupled to cob(II)alamin whenever reaction mixtures with ethanolamine or with (R) or (S)-2-aminopropanol are frozen during turnover (15-18). EPR spectra of the paramagnetic species consist of the gx and gy signals of cob(II)alamin at g ∼ 2.3 and the signal of the organic radicals at g ∼ 2.003 (17). Cob(II)alamin and the organic radicals interact magnetically through exchange and dipole-dipole interactions, creating spectra in which signals for each paramagnetic species are perturbed by the electron-electron spin coupling with the partner (19). The EPR spectrum of a radical intermediate in the reaction of EAL with ethanolamine was previously assigned as a product-like carbinolamine having unpaired spin on C2 (20). The distance between the radical intermediate and the cobalt center of cob(II)alamin was determined to be ∼ 9.7 A (21). Theoretical hyperfine splitting values of different possible radical identities and conformations were obtained using electronic structure calculations (22). The structure of a radical intermediate of ethanolamine was further characterized by electron spin echo envelope modulation spectroscopy (13, 23, 24). However, conclusions from all of these studies were based on the original assignment of the radical intermediate as the carbinolamine product radical (20). In the earlier studies, concentrated solutions of enzyme were pre-mixed with a concentrated solution of ethanolamine (20). The reaction was initiated by addition of AdoCbl to the enzyme/substrate mixture prior to manual freezing of the reaction mixtures. There is a slow binding of AdoCbl to the enzyme that delays consumption of substrate using this order-of-addition (25, 26). AdoCbl binds to the enzyme with high affinity, and its binding and dissociation are probably not part of the normal catalytic cycle (27). Moreover, significant amounts of acetaldehyde and ammonia are generated during the several seconds required to mix and freeze the sample. In this study, the radical intermediate observed during the steady-state regime in the reaction of EAL with ethanolamine is characterized by RMFQ trapping methods (28). These methods make it possible to use much lower concentrations of substrate and buffer and to use the physiological order-of-addition of substrate to the enzyme-cofactor complex. The radical intermediates are probed throughout the time-course of substrate turnover with C1 and C2 isotopomers of (13C)ethanolamine. Distance and geometrical information regarding the positions of the radical relative to cob(II)alamin are obtained from detailed analysis of electron spin-spin and nuclear spin electron spin interactions.