Dataset Information

Entropic origin of cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase.

ABSTRACT: Adenosylcobalamin-dependent enzymes accelerate the cleavage of the cobalt-carbon (Co-C) bond of the bound coenzyme by >10(10)-fold. The cleavage-generated 5'-deoxyadenosyl radical initiates the catalytic cycle by abstracting a hydrogen atom from substrate. Kinetic coupling of the Co-C bond cleavage and hydrogen-atom-transfer steps at ambient temperatures has interfered with past experimental attempts to directly address the factors that govern Co-C bond cleavage catalysis. Here, we use time-resolved, full-spectrum electron paramagnetic resonance spectroscopy, with temperature-step reaction initiation, starting from the enzyme-coenzyme-substrate ternary complex and (2)H-labeled substrate, to study radical pair generation in ethanolamine ammonia-lyase from Salmonella typhimurium at 234-248 K in a dimethylsulfoxide/water cryosolvent system. The monoexponential kinetics of formation of the (2)H- and (1)H-substituted substrate radicals are the same, indicating that Co-C bond cleavage rate-limits radical pair formation. Analysis of the kinetics by using a linear, three-state model allows extraction of the microscopic rate constant for Co-C bond cleavage. Eyring analysis reveals that the activation enthalpy for Co-C bond cleavage is 32 ± 1 kcal/mol, which is the same as for the cleavage reaction in solution. The origin of Co-C bond cleavage catalysis in the enzyme is, therefore, the large, favorable activation entropy of 61 ± 6 cal/(mol·K) (relative to 7 ± 1 cal/(mol·K) in solution). This represents a paradigm shift from traditional, enthalpy-based mechanisms that have been proposed for Co-C bond-breaking in B12 enzymes. The catalysis is proposed to arise from an increase in protein configurational entropy along the reaction coordinate.

SUBMITTER: Wang M

PROVIDER: S-EPMC3839591 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Entropic origin of cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase.

Wang Miao M Warncke Kurt K

Journal of the American Chemical Society 20131001 40

Adenosylcobalamin-dependent enzymes accelerate the cleavage of the cobalt-carbon (Co-C) bond of the bound coenzyme by >10(10)-fold. The cleavage-generated 5'-deoxyadenosyl radical initiates the catalytic cycle by abstracting a hydrogen atom from substrate. Kinetic coupling of the Co-C bond cleavage and hydrogen-atom-transfer steps at ambient temperatures has interfered with past experimental attempts to directly address the factors that govern Co-C bond cleavage catalysis. Here, we use time-reso ...[more]

PMID: 24028405

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Project description:Protein contributions to the substrate-triggered cleavage of the cobalt-carbon (Co-C) bond and formation of the cob(II)alamin-5'-deoxyadenosyl radical pair in the adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been studied by using pulsed-laser photolysis of AdoCbl in the EAL-AdoCbl-substrate ternary complex, and time-resolved probing of the photoproduct dynamics by using ultraviolet-visible absorption spectroscopy on the 10(-7)-10(-1) s time scale. Experiments were performed in a fluid dimethylsulfoxide/water cryosolvent system at 240 K, under conditions of kinetic competence for thermal cleavage of the Co-C bond in the ternary complex. The static ultraviolet-visible absorption spectra of holo-EAL and ternary complex are comparable, indicating that the binding of substrate does not labilize the cofactor cobalt-carbon (Co-C) bond by significantly distorting the equilibrium AdoCbl structure. Photolysis of AdoCbl in EAL at 240 K leads to cob(II)alamin-5'-deoxyadenosyl radical pair quantum yields of <0.01 at 10(-6) s in both holo-EAL and ternary complex. Three photoproduct states are populated following a saturating laser pulse, and labeled, P(f), P(s), and P(c). The relative amplitudes and first-order recombination rate constants of P(f) (0.4-0.6; 40-50 s(-1)), P(s) (0.3-0.4; 4 s(-1)), and P(c) (0.1-0.2; 0) are comparable in holo-EAL and in the ternary complex. Time-resolved, full-spectrum electron paramagnetic resonance (EPR) spectroscopy shows that visible irradiation alters neither the kinetics of thermal cob(II)alamin-substrate radical pair formation, nor the equilibrium between ternary complex and cob(II)alamin-substrate radical pair, at 246 K. The results indicate that substrate binding to holo-EAL does not "switch" the protein to a new structural state, which promptly stabilizes the cob(II)alamin-5'-deoxyadenosyl radical pair photoproduct, either through an increased barrier to recombination, a decreased barrier to further radical pair separation, or lowering of the radical pair state free energy, or a combination of these effects. Therefore, we conclude that such a change in protein structure, which is independent of changes in the AdoCbl structure, and specifically the Co-C bond length, is not a basis of Co-C bond cleavage catalysis. The results suggest that, following the substrate trigger, the protein interacts with the cofactor to contiguously guide the cleavage of the Co-C bond, at every step along the cleavage coordinate, starting from the equilibrium configuration of the ternary complex. The cleavage is thus represented by a diagonal trajectory across a free energy surface, that is defined by chemical (Co-C separation) and protein configuration coordinates.

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.

Project description:The quantum yield and kinetics of decay of cob(II)alamin formed by pulsed-laser photolysis of adenosylcobalamin (AdoCbl; coenzyme B(12)) in AdoCbl-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been studied on the 10(-7)-10(-1) s time scale at 295 K by using transient ultraviolet-visible absorption spectroscopy. The aim is to probe the mechanism of formation and stabilization of the cob(II)alamin-5'-deoxyadenosyl radical pair, which is a key intermediate in EAL catalysis, and the influence of substrate binding on this process. Substrate binding is required for cobalt-carbon bond cleavage in the native system. Photolysis of AdoCbl in EAL leads to a quantum yield at 10(-7) s for cob(II)alamin of 0.08 +/- 0.01, which is 3-fold smaller than for AdoCbl in aqueous solution (0.23 +/- 0.01). The protein binding site therefore suppresses photoproduct radical pair formation. Three photoproduct states, P(f), P(s), and P(c), are identified in holo-EAL by the different cob(II)alamin decay kinetics (subscripts denote fast, slow, and constant, respectively). These states have the following first-order decay rate constants and quantum yields: 2.2 x 10(3) s(-1) and 0.02 for P(f), 4.2 x 10(2) s(-1) and 0.01 for P(s), and constant amplitude, with no recombination, and 0.05 for P(c), respectively. Binding of the substrate analogue (S)-1-amino-2-propanol to EAL eliminates the P(f) state and lowers the quantum yield of P(c) (0.03) relative to that of P(s) (0.01) but does not significantly change the quantum yield or decay rate constant of P(s), relative to those of holo-EAL. The substrate analogue thus influences the quantum yield at 10(-7) s by changing the cage escape rate from the geminate cob(II)alamin-5'-deoxyadenosyl radical pair state. However, the predicted substrate analogue binding-induced increase in the quantum yield is not observed. It is proposed that the substrate analogue does not induce the radical pair stabilizing changes in the protein that are characteristic of true substrates.

Dataset Information

Entropic origin of cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase.

Publications

Entropic origin of cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase.

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