Probing Nitrogen-Sensitive Steps in the Free-Radical-Mediated Deamination of Amino Alcohols by Ethanolamine Ammonia-Lyase

Abstract

The contribution of C-N bond-breaking/making steps to the rate of the free-radical-mediated deamination of vicinal amino alcohols by adenosylcobalamin-dependent ethanolamine ammonialyase has been investigated by 15 N isotope effects (IE's) and by electron paramagnetic resonance (EPR) spectroscopy. 15 N IE's were determined for three substrates, ethanolamine, (R)-2-aminopropanol, and (S)-2-aminopropanol using isotope ratio mass spectrometry analysis of the product ammonia. Measurements with all three substrates gave measurable, normal 15 N IE's; however, the IE of (S)-2-aminopropanol was ∼ 5-fold greater than the other two. Reaction mixtures frozen during the steady-state show that the 2-aminopropanols give EPR spectra characteristic of the initial substrate radical whereas ethanolamine gives spectra consistent with a product-related radical [Warncke, K.; Schmidt, J. C.; Kee, S.-C., J. Am. Chem. Soc. 1999, 121, 10522-10528]. The steadystate concentration of the radical with (R)-2-aminopropanol is ∼ half that observed with the S isomer, and with (R)-2-aminopropanol the steady-state level of radical is further reduced upon deuteration at C1. The results show that relative heights of kinetic barriers differ among the three substrates such that levels or identities of steady-state intermediates differ. 15 N-Sensitive steps are significant contributors to V/K with (S)-2-aminopropanol.The bacterial enzyme, ethanolamine ammonia-lyase (EAL, EC 4.3.1.7) catalyzes the adenosylcobalamin (AdoCbl)-dependent deamination of ethanolamine or 2-aminopropanols to ammonia and the corresponding aldehyde. 1 The reaction is one of several 1-2 radicalrearrangements/eliminations catalyzed by AdoCbl-dependent enzymes. 2-4 The paradigm for these reactions is an interchange of a group at C2 and a hydrogen atom at C1. With the exception of glutamate mutase, 5 mechanistic details of the migration steps in the AdoCbl-dependent enzymes are not well understood. 3 The EAL reaction can be considered as a C2 → C1 migration of ammonia, followed by decomposition of the product carbinolamine (Scheme 1). 1Migration of ammonia to C1 is not strictly required to form acetaldehyde. 6 The migration mechanism has been demonstrated in the related enzyme dioldehydrase, and derives some support from stereochemistry. 7 Computational studies have indicated that both an internal migration of ammonia or direct elimination are energetically feasible. 6, 8The mechanism includes two H atom abstraction steps that are energetically demanding. Isotopes of H have been used to determine the contributions of these H atom transfers to the overall rate. 1 Transfer of 3 H from 5′ position of AdoCbl to acetaldehyde during turnover shows an isotope effect (IE) of ∼100. ]-ethanolamine is ∼ 6. 9, 13 For (S)-and (R)-2-aminopropanol, D V's are both ∼5. 14 Attenuation of the 2 H IE in the V max suggests that H-insensitive steps in the reaction contribute to limiting of the rate. For example, V/K for the EAL reaction is sensitive to external magnetic fields, suggesting that ra...

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

“…In the case of EAL, the k cat for steady-state turnover and the room-temperature rate constant for decay of the steady-state (corresponding to the accumulated Co 2+ -substrate radical pair state) following substrate depletion are comparable (Bandarian & Reed, 2000; Hollaway et al, 1978), and values of k cat , measured at 277 and 295 K, adhere to the linear extrapolation of the monotonic ln k versus T −1 data for 220–230 K (C. Zhu, M. Kohne, K. Warncke, unpublished). This is consistent with rate-determination by the radical rearrangement reaction from room- to low-tempeature, and with the partial substrate 14 N/ 15 N steady-state isotope effect, that is assigned to the substrate C-N bond cleavage sub-step (Frey, 2010; Poyner, Anderson, Bandarian, Cleland, & Reed, 2006). …”

Kinetic Isolation and Characterization of the Radical Rearrangement Step in Coenzyme B12-Dependent Ethanolamine Ammonia-lyase

“…Consistent with this observation, the 1 H-aminoethanol substrate 14 N/ 15 N steady-state kinetic IE on V / K M ( V , maximum velocity; K M , Michaelis constant) and V was proposed to arise from C2-N bond cleavage in the rearrangement step 12,19. However, the relatively low value of the 15 N-kinetic IE led to the proposal that hydrogen atom transfer was largely rate limiting in the steady-state reaction of 1 H-aminoethanol substrate 20.…”

“…The accumulation of the Co II -aminoethanol substrate radical pair as the only paramagnetic intermediate under steady-state turnover conditions,12 and the substrate 14 N/ 15 N IE on V / K M and V ,12,19 indicate that the rearrangement step presents a significant barrier to the overall steady-state reaction sequence, for the 1 H-aminoethanol substrate,12 but the relatively small value of the 15 N-kinetic IE (0.17%)19 led to the proposal that hydrogen transfer is largely rate limiting for the steady-state reaction at room temperature 20. In contrast, the results and interpretation, presented here, establish that the radical rearrangement is the dominant rate-determining step in the decay reaction of the cryotrapped substrate radical at 190-207 K. The low temperature results lead to the prediction that the rate constant for the HT2 step is temperature dependent ( k HT decreases with decreasing temperature), because the rate constant for radical rearrangement is relatively temperature independent (small activation entropy).…”

Two Dynamical Regimes of the Substrate Radical Rearrangement Reaction in B12-Dependent Ethanolamine Ammonia-Lyase Resolve Contributions of Native Protein Configurations and Collective Configurational Fluctuations to Catalysis

“…49,50 The equivalence of k cat and the substrate radical decay rate constant is congruent with rearrangement as the dominant internal step in rate determination of turnover. 32,33 Therefore, we measured k cat values of EAL for aminoethanol at 277 K [5.7 (±0.4) × 10° s −1 ] and 295 K [2.9 (±0.2) × 10 1 s −1 ] (mean value, ± standard deviation, n = 3).…”