Adenosylcobalamin-dependent Ethanolamine Ammonia-lyase Research Articles

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.

Approaches to the resolution and characterization of individual chemical steps in enzyme catalytic sequences, by using temperatures in the cryogenic range of 190-250 K, and kinetics measured by time-resolved, full-spectrum electron paramagnetic resonance spectroscopy in fluid cryosolvent and frozen solution systems, are described. The preparation and performance of the adenosylcobalamin-dependent ethanolamine ammonia-lyase enzyme from Salmonella typhimurium in the two systems exemplifies the biochemical and spectroscopic methods. General advantages of low-temperature studies are (1) slowing of reaction steps, so that measurements can be made by using straightforward T-step kinetic methods and commercial instrumentation, (2) resolution of individual reaction steps, so that first-order kinetic analysis can be applied, and (3) accumulation of intermediates that are not detectable at room temperatures. The broad temperature range from room temperature to 190 K encompasses three regimes: (1) temperature-independent mean free energy surface (corresponding to native behavior); (2) the narrow temperature region of a glass-like transition in the protein, over which the free energy surface changes, revealing dependence of the native reaction on collective protein/solvent motions; and (3) the temperature range below the glass transition region, for which persistent reaction corresponds to nonnative, alternative reaction pathways, in the vicinity of the native configurational envelope. Representative outcomes of low-temperature kinetics studies are portrayed on Eyring and free energy surface (landscape) plots, and guidelines for interpretations are presented.

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

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.

Cobinamide production of hydrogen in a homogeneous aqueous photochemical system, and assembly and photoreduction in a (βα)8 protein.

Components of a protein-integrated, earth-abundant metal macrocycle catalyst, with the purpose of H2 production from aqueous protons under green conditions, are characterized. The cobalt-corrin complex, cobinamide, is demonstrated to produce H2 (4.4±1.8×10(-3) turnover number per hour) in a homogeneous, photosensitizer/sacrificial electron donor system in pure water at neutral pH. Turnover is proposed to be limited by the relatively low population of the gateway cobalt(III) hydride species. A heterolytic mechanism for H2 production from the cobalt(II) hydride is proposed. Two essential requirements for assembly of a functional protein-catalyst complex are demonstrated for interaction of cobinamide with the (βα)8 TIM barrel protein, EutB, from the adenosylcobalamin-dependent ethanolamine ammonia lyase from Salmonella typhimurium: (1) high-affinity equilibrium binding of the cobinamide (dissociation constant 2.1×10(-7)M) and (2) in situ photoreduction of the cobinamide-protein complex to the Co(I) state. Molecular modeling of the cobinamide-EutB interaction shows that these features arise from specific hydrogen-bond and apolar interactions of the protein with the alkylamide substituents and the ring of the corrin, and accessibility of the binding site to the solution. The results establish cobinamide-EutB as a platform for design and engineering of a robust H2 production metallocatalyst that operates under green conditions and uses the advantages of the protein as a tunable medium and material support.

Characterization of protein contributions to cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase by using photolysis in the ternary complex.

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.

Magnetic Field Effect Studies Indicate Reduced Geminate Recombination of the Radical Pair in Substrate-Bound Adenosylcobalamin-Dependent Ethanolamine Ammonia Lyase

The apparent conflict between literature evidence for (i) radical pair (RP) stabilization in adenosylcobalamin (AdoCbl)-dependent enzymes and (ii) the manifestation of magnetic field sensitivity due to appreciable geminate recombination of the RP has been reconciled by pre-steady-state magnetic field effect (MFE) investigations with ethanolamine ammonia lyase (EAL). We have shown previous stopped-flow MFE studies to be insensitive to magnetically induced changes in the net forward rate of C-Co homolytic bond cleavage. Subsequently, we observed a magnetic-dependence in the continuous-wave C-Co photolysis of free AdoCbl in 75% glycerol but have not done so in the thermal homolysis of this bond in the enzyme-bound cofactor in the presence of substrate. Consequently, in the enzyme-bound state, the RP generated upon homolysis appears to be stabilized against the extent of geminate recombination required to observe an MFE. These findings have strong implications for the mechanism of RP stabilization and the unprecedented catalytic power of this important class of cobalamin-dependent enzymes.

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

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 ammonia-lyase has been investigated by 15N isotope effects (IE's) and by electron paramagnetic resonance (EPR) spectroscopy. 15N 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 15N IE's; however, the IE of (S)-2-aminopropanol was approximately 5-fold greater than that of 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 steady-state concentration of the radical with (R)-2-aminopropanol is about half that observed with the S isomer, and with (R)-2-aminopropanol, the steady-state level of the 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. 15N-sensitive steps are significant contributors to V/K with (S)-2-aminopropanol.

Identification of a reactivating factor for adenosylcobalamin-dependent ethanolamine ammonia lyase.

The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+, and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5' end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O2-inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg2+. The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.

Stereochemistry of the rearrangement of 2-aminoethanol by ethanolamine ammonia-lyase.

(1R)-2-Amino[1-2H1]ethanol and (1S,2RS)-2-amino[1,2-2H2]ethanols have been synthesised by decarboxylation of (2S,3R)-[3-2H1]serine and (2S,3S)-[2,3-2H2]serine respectively. The stereochemical integrity of these labelled 2-aminoethanols has been ascertained from the 1H-NMR spectra of their N,O-dicamphanoyl derivatives. This assay has also been used to confirm that samples of (2R)- and (2S)-2-amino [2-2H1]ethanols prepared from (2R)- and (2S)-[2-2H1]glycines are stereochemically pure. Ethanolamine ammonia-lyase rearranges (1R)-2-amino[1-2H1]ethanol to acetaldehyde at approximately the same rate as it rearranges unlabelled 2-aminoethanol, whilst (1S,2RS)-2-amino[1,2-2H2]ethanol is rearranged at the same rate as the (1,1-2H2)-labelled substrate. The isotope effect is approximately kH/k2H = 8. The 2H-NMR spectra of the 3,5-dinitrobenzoates of the ethanol produced by reduction in situ of the acetaldehyde formed in the rearrangements show that the 1-2H1 label migrates in (1S,2RS)-2-amino-[1,2-2H2]ethanol and 2-amino[1,1-2H2]ethanol but not in (1R)-2-amino[1-2H1]ethanol. The above results indicate that the adenosylcobalamin-dependent ethanolamine ammonia-lyase catalyses the rearrangement of 2-aminoethanol with migration of the 1-pro-S-hydrogen atom.

Regulatory and structural interrelationships between adenosylcobalamin-dependent ethanolamine ammonia-lyase and the CoA-dependent aldehyde dehydrogenases of Escherichia coli

Escherichia coli metabolizes ethanolamine to acetyl CoA via acetaldehyde (Shukla & Turner, 1980). The deamination step is catalysed by cobamide-dependent ethanolamine ammonia-lyase (EAL; EC 4.3.1.7), and the resulting acetaldehyde is further metabolized by CoA-dependent acetaldehyde dehydrogenase (ALDH) (EC 1.2.1.10). EAL is composed of six large structural or regulatory subunits (protein I, mo1.W. 56 900) and six small catalytic subunits (protein 11, mo1.W. 35 200) (Pennington et al., 1981). Induction of EAL requires the concerted action of both ethanolamine and coenzyme B,, (Blackwell & Turner, 1978). Cultures of E. coli type-I (N.C.I.B. 8114) and its derived mutants, N.C.I.B. 11361 and PDT 31, were supplied with nitrogen as either ethanolamine (1 ghitre) plus vitamin B,, (4O,ug/litre), i.e. inducing conditions, or (NH,),SO, (1 ghitre), i.e. non-inducing conditions. Elution from Bio-Gel A-1.5 m of cell-free extracts from type-I cultures grown under inducing conditions produced a peak with both EAL and ALDH activity at an elution volume corresponding to a 'spherical' mo1.W. of 520000 (ALDH isoenzyme A) with a second ALDH peak at 370000 (isoenzyme B). The two co-eluting enzyme activities were separable by anion-exchange chromatography on DEAEcellulose. After this latter step, it was found that the molecular weight of isoenzyme A had fallen to 12OOO0, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that this enzyme was a dimer of subunit mol.wt. 58 100. Extracts from E. coli type-I cultures grown under non-inducing conditions contained no EAL activity but significant ALDH activity

A spectrophotometric rapid kinetic study of reactions catalysed by coenzyme-B12-dependent ethanolamine ammonia-lyase.

Spectral changes during the transient states and the steady states of the reactions catalysed by the adenosylcobalamin‐dependent ethanolamine ammonia‐lyase with l‐2‐aminopropanol and 2‐aminoethanol as substrates were followed by using a rapid‐wavelength‐scanning, stopped‐flow spectrophotometer (800 spectra per second and each spectrum 340–560 nm). During the steady state of the reaction with the ‘poor’ substrate, l‐2‐aminopropanol (kcat∼ 1 s−1), under conditions of V, the spectrum of the coenzyme closely resembled that of cob(II)alamin. With the ‘good’ substrate, 2‐aminoethanol (kcat∼ 140 s−1), under the same conditions, the spectrum corresponded to a mixture of 58% cob(II)alamin and 42% cob(III)alamin. The latter may be either unchanged adenosylcobalamin or a coenzyme derivative with a covalent bond between a substrate carbon and the cobalt atom. The magnitude of a reported kinetic deuterium isotope effect favours the second possibility. There was no evidence for the presence of a cob(I)alamin intermediate. The transient phases of the reactions were dependent on the order of mixing of reactants. When enzyme was placed in one syringe of the stopped‐flow apparatus and coenzyme and substrate in the other, the cob(II)alamin intermediate formed slowly (first‐order rate constants of about 5 s−1 with l‐2‐aminopropanol as substrate and about 2 s−1 with 2‐aminoethanol). When the enzyme and coenzyme were placed in one syringe and the substrate in the other, the formation of the cob(II)alamin occurred much more rapidly (90 s−1 with l‐2‐aminopropanol and greater than 300 s−1 with 2‐aminoethanol). It is concluded that binding of adenosylcobalamin to the enzyme is followed by a slow change in the conformation of the enzyme molecule to give a catalytically active species (there is no observable change in the spectrum of the coenzyme concommitant with this process). The formation of active holoenzyme is slow on the time scale of subsequent catalytic steps when 2‐aminoethanol is the substrate. The spectra of the intermediates formed in the reactions with both substrates, the rates of their formation and their lifetimes allowed a detailed kinetic analysis of the reactions in terms of a three‐step mechanism involving binding of substrate, cob(II)alamin formation (k+2 step) and cob(II)alamin breakdown (k+3 step). In catalysis the difference between the substrates is expressed mainly in the k+3 step (240 s−1 and about 1 s−1 respectively for 2‐aminoethanol and l‐2‐aminopropanol, the respective k+2 values being 336 s−1 and 90 s−1).

The Stereochemistry of the Reaction Catalyzed by Ethanolamine AmmoniaLyase, an Adenosylcobalamindependent Enzyme: AN EXAMPLE OF RACEMIZATION ACCOMPANYING SUBSTITUTION

Abstract Upon deamination of chirally labeled [2-2H, 3H]ethanolamine by the adenosylcobalamin-dependent ethanolamine ammonia-lyase (EC 4.3.1.7) from Clostridium sp., chirality of the labeled carbon atom is lost. This result is consistent with a mechanism involving the 1-amino-1-hydroxyethane2-yl radical as a catalytic intermediate.