Is There a Dynamic Protein Contribution to the Substrate Trigger in Coenzyme B<sub>12</sub>‐Dependent Ethanolamine Ammonia Lyase?

Jones, Alex R.; Hardman, Samantha J. O.; Hay, Sam; Scrutton, Nigel S.

doi:10.1002/ange.201105132

Cited by 13 publications

(28 citation statements)

References 43 publications

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“…The configurational catalysis model is consistent with the low quantum yield (<10 –2 ) of Co(II)-radical pair formation observed in transient photolysis studies of AdoCbl in the EAL ternary complex at 240 K, 16 changes in EAL protein secondary structure following Co-C bond thermolysis detected by infrared spectroscopy, 58 and the solution viscosity-dependence of the Co(II)-radical pair lifetime in EAL. 59 …”

Section: Discussionmentioning

confidence: 99%

Entropic Origin of Cobalt–Carbon Bond Cleavage Catalysis in Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase

Wang

Warncke

2013

J. Am. Chem. Soc.

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Adenosylcobalamin-dependent enzymes accelerate the cleavage of the cobalt-carbon (Co-C) bond of the bound coenzyme by >1011-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, temperature-step reaction initiation, starting from the enzyme-coenzyme-substrate ternary complex, and 2H-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 2H- and 1H-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.

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Section: Discussionmentioning

confidence: 99%

Entropic Origin of Cobalt–Carbon Bond Cleavage Catalysis in Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase

Wang

Warncke

2013

J. Am. Chem. Soc.

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“…Table 4. individual reaction steps in enzyme catalysis [17,[42][43][44]. The viscosity measurements indicate that protein motion is coupled kinetically to the rate of C-Co bond homolysis in both wild-type and variant forms of OAM.…”

Section: Enzymementioning

confidence: 97%

Glutamate 338 is an electrostatic facilitator of C–Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase

Menon

Fisher

et al. 2015

The FEBS Journal

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How cobalamin-dependent enzymes promote C–Co homolysis to initiate radical catalysis has been debated extensively. For the pyridoxal 5′-phosphate and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5-aminomutase (OAM), large-scale re-orientation of the cobalamin-binding domain linked to C–Co bond breakage has been proposed. In these models, substrate binding triggers dynamic sampling of the B12-binding Rossmann domain to achieve a catalytically competent ‘closed’ conformational state. In ‘closed’ conformations of OAM, Glu338 is thought to facilitate C–Co bond breakage by close association with the cobalamin adenosyl group. We investigated this using stopped-flow continuous-wave photolysis, viscosity dependence kinetic measurements, and electron paramagnetic resonance spectroscopy of a series of Glu338 variants. We found that substrate-induced C–Co bond homolysis is compromised in Glu388 variant forms of OAM, although photolysis of the C–Co bond is not affected by the identity of residue 338. Electrostatic interactions of Glu338 with the 5′-deoxyadenosyl group of B12 potentiate C–Co bond homolysis in ‘closed’ conformations only; these conformations are unlocked by substrate binding. Our studies extend earlier models that identified a requirement for large-scale motion of the cobalamin domain. Our findings indicate that large-scale motion is required to pre-organize the active site by enabling transient formation of ‘closed’ conformations of OAM. In ‘closed’ conformations, Glu338 interacts with the 5′-deoxyadenosyl group of cobalamin. This interaction is required to potentiate C–Co homolysis, and is a crucial component of the approximately 1012 rate enhancement achieved by cobalamin-dependent enzymes for C–Co bond homolysis.

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“…First, AdoCbl photolysis creates the same RP. The photophysical and photochemical dynamics of both unbound [17,18,[54][55][56][57][58][59] and enzyme-bound [60][61][62][63][64] AdoCbl have been well characterized, allowing us to accurately assess any perturbation of these dynamics from the protein. After excitation in water, there is rapid relaxation from the excited state(s) to a cob(II) alamin-like species (Fig.…”

Section: Coenzyme B 12 -Dependent Enzymology: a Physical Chemistry Pementioning

confidence: 99%

“…In water, the majority (75-80%) of photo-generated 'close' RPs recombine within approximately 10 ns, the quantum yield of separated radicals being controlled by competition between geminate recombination and cage escape [17,[54][55][56]. Due to this 'cage' effect, both increased solvent viscosity and binding to an enzyme shift the balance even further towards geminate recombination (90-95%) [56,[60][61][62]64].…”

Section: Coenzyme B 12 -Dependent Enzymology: a Physical Chemistry Pementioning

confidence: 99%

“…Recent studies exploiting the photo-and spin chemistry of AdoCbl, together with time-resolved vibrational methods, suggest there may also be a subtle dynamic contribution from this and other activesite residues in EAL [59,62,63,77]. Continuous-wave photolysis MFE data for both free and EAL-bound AdoCbl in the absence of substrate show compelling viscosity dependencies that provide information on the possible time scales of some of these motions [62]. As expected, the photolysis rate (k obs ) of free AdoCbl and the magnitude of MFE on k obs showed a decrease and an increase, respectively, with increasing solvent viscosity.…”

Section: Small-scale Motions and Localized Effectsmentioning

confidence: 99%

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Relating localized protein motions to the reaction coordinate in coenzyme B₁₂‐dependent enzymes

et al. 2013

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The classical picture of enzyme catalysis relies on controlling the entropic and enthalpic contributions by manipulating reaction barriers and co-locating reactants and cofactors to facilitate the reaction chemistry. Catalysis is linked inextricably to the geometry of the enzyme-substrate complex and the chemical/physical properties of the active site, and probably to dynamical contributions that guide reactants along the desired reaction coordinate. Coenzyme B 12 -dependent enzymes have remarkable catalytic power and unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. Here we discuss recent developments that are beginning to provide atomistic insight into how coenzyme B 12 -dependent enzymes steer reactants along the reaction coordinate. Such insight will ultimately generate 'movies' of the catalytic process across all relevant time scales. In the longer term, this will enable more predictive engineering of this class of enzyme to achieve new and desirable chemical outcomes.

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Is There a Dynamic Protein Contribution to the Substrate Trigger in Coenzyme B₁₂‐Dependent Ethanolamine Ammonia Lyase?

Cited by 13 publications

References 43 publications

Entropic Origin of Cobalt–Carbon Bond Cleavage Catalysis in Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase

Entropic Origin of Cobalt–Carbon Bond Cleavage Catalysis in Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase

Glutamate 338 is an electrostatic facilitator of C–Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase

Relating localized protein motions to the reaction coordinate in coenzyme B₁₂‐dependent enzymes

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Is There a Dynamic Protein Contribution to the Substrate Trigger in Coenzyme B12‐Dependent Ethanolamine Ammonia Lyase?

Cited by 13 publications

References 43 publications

Entropic Origin of Cobalt–Carbon Bond Cleavage Catalysis in Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase

Entropic Origin of Cobalt–Carbon Bond Cleavage Catalysis in Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase

Glutamate 338 is an electrostatic facilitator of C–Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase

Relating localized protein motions to the reaction coordinate in coenzyme B12‐dependent enzymes

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Product

Resources

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Is There a Dynamic Protein Contribution to the Substrate Trigger in Coenzyme B₁₂‐Dependent Ethanolamine Ammonia Lyase?

Relating localized protein motions to the reaction coordinate in coenzyme B₁₂‐dependent enzymes