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Probing reversible chemistry in coenzyme B12 -dependent ethanolamine ammonia lyase with kinetic isotope effects.

ABSTRACT: Coenzyme B12 -dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the ?-carbon of the 5'-deoxyadenosyl moiety of the intrinsic coenzyme B12 , it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5'-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5'-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with Co?C homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small.

SUBMITTER: Jones AR

PROVIDER: S-EPMC4497352 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Probing reversible chemistry in coenzyme B12 -dependent ethanolamine ammonia lyase with kinetic isotope effects.

Jones Alex R AR Rentergent Julius J Scrutton Nigel S NS Hay Sam S

Chemistry (Weinheim an der Bergstrasse, Germany) 20150507 24

Coenzyme B12 -dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the β-carbon of the 5'-deoxyadenosyl moiety of the intrinsic coenzyme B12 , it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5'-deoxyadenosyl radical and substrate during sin ...[more]

PMID: 25950663

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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.

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.

Dataset Information

Probing reversible chemistry in coenzyme B12 -dependent ethanolamine ammonia lyase with kinetic isotope effects.

Publications

Probing reversible chemistry in coenzyme B12 -dependent ethanolamine ammonia lyase with kinetic isotope effects.

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