Ethanolamine Deaminase Research Articles

Identification of a Rearranged-Substrate, Product Radical Intermediate and the Contribution of a Product Radical Trap in Vitamin B12 Coenzyme-Dependent Ethanolamine Deaminase Catalysis

The radical intermediate present during steady-state turnover of substrate aminoethanol by ethanolamine deaminase from Salmonella typhimurium has been characterized by using X-band electron paramag...

Characterization of the Product Radical Structure in the CoII-Product Radical Pair State of Coenzyme B12-Dependent Ethanolamine Deaminase by Using Three-Pulse2H ESEEM Spectroscopy

Molecular structural features of the product radical in the Co(II)-product radical pair catalytic intermediate state in coenzyme B(12)- (adenosylcobalamin-) dependent ethanolamine deaminase from Salmonella typhimurium have been characterized by using X-band three-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy in the disordered solid state. The Co(II)-product radical pair state was prepared by cryotrapping holoenzyme during steady-state turnover on excess 1,1,2,2-(2)H(4)-aminoethanol or natural abundance, (1)H(4)-aminoethanol. Simulation of the (2)H/(1)H quotient ESEEM (obtained at two microwave frequencies, 8.9 and 10.9 GHz) from the interaction of the unpaired electron localized at C2 of the product radical with nearby (2)H nuclei requires four types of coupled (2)H, which are assigned as follows: (a) a single strongly coupled (effective dipole distance, r(eff) = 2.3 A) (2)H in the C5' methyl group of 5'-deoxyadenosine, (b) two weakly coupled (r(eff) = 4.2 A) (2)H in the C5' methyl group, (c) one (2)H coupling from a beta-(2)H bonded to C1 of the product radical (isotropic hyperfine coupling, A(iso) = 4.7 MHz), and (d) a second type of C1 beta-(2)H coupling (A(iso) = 7.7 MHz). The two beta-(2)H couplings are proposed to arise from two C1-C2 rotamer states of the product radical that are present in approximately equal proportion. A model is presented, in which C5' is positioned at a distance of 3.3 A from C2, which is comparable with the C1-C5' distance in the Co(II)-substrate radical pair intermediate. Therefore, the C5'methyl group remains in close (van der Waals) contact with the substrate and product radical species during the radical rearrangement step of the catalytic cycle, and the C5' center is the sole mediator of radical pair recombination in ethanolamine deaminase.

Active Site Reactant Center Geometry in the CoII−Product Radical Pair State of Coenzyme B12-Dependent Ethanolamine Deaminase Determined by Using Orientation-Selection Electron Spin−Echo Envelope Modulation Spectroscopy

The distances and orientations among reactant centers in the active site of coenzyme B12-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized in the Co(II)-product radical pair state by using X-band electron paramagnetic resonance (EPR) and two-pulse electron spin-echo envelope modulation (ESEEM) spectroscopies in the disordered solid state. The unpaired electron spin in the product radical is localized on C2. Our approach is based on the orientation-selection created in the EPR spectrum of the biradical by the axial electron-electron dipolar interaction. Simulation of the EPR line shape yielded a best-fit Co(II)-C2 distance of 9.3 A. ESEEM spectroscopy performed at four magnetic field values addressed the hyperfine coupling of the unpaired electron spin on C2 with 2H in the C5' methyl group of 5'-deoxyadenosine and in the beta-2H position at C1 of the radical. Global ESEEM simulations (over the four magnetic fields) were weighted by the orientation dependence of the EPR line shape. A Nelder-Mead direct search fitting algorithm was used to optimize the simulations. The results lead to a partial model of the active site, in which C5' is located a perpendicular distance of 1.6 A from the Co(II)-C2 axis, at distances of 6.3 and 3.5 A from Co(II) and C2, respectively. The van der Waals contact of the C5'-methyl group and C2 indicates that C5' remains close to the radical species during the rearrangement step. The C2-Hs-C5' angle including the strongly coupled hydrogen, Hs, and the C5'-Hs orientation relative to the C1-C2 axis are consistent with a linear hydrogen atom transfer coordinate and an in-line acceptor p-orbital orientation. The trigonal plane of the C2 atom defines sub-spaces within the active site for C5' radical migration and hydrogen atom transfers (side of the plane facing Co(II)) and amine migration (side of the plane facing away from Co(II)).

Molecular properties of the product radical in adenosylcobalamin-dependent ethanolamine deaminase

The adenosylcobalamin coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium catalyzes the deamination of aminoethanol to ethanol and ammonia. The product radical observed during steady-state turnover of substrate aminoethanol has been characterized by electron paramagnetic resonance technique. This study explores the conformational dependent hyperfine coupling constants and energetics of the possible product radical intermediates by means of density functional theory based calculations; the results are compared with experimental ones derived from EPR spectra simulations. We have obtained sets of possible conformational structures of the observed product radical indicating that the radical trapped during the catalysis of ethanolamine deaminase corresponds to an activated energy state facilitating the subsequent hydrogen atom abstraction from the inert 5 ′ -methyl group of deoxyadenosine.

Direct determination of product radical structure reveals the radical rearrangement pathway in a coenzyme B12-dependent enzyme.

A carbinolamine (1-aminoethan-1-ol-2-yl) structure for the product radical in the CoII product radical pair catalytic intermediate state in coenzyme B12 (adenosylcobalamin)-dependent ethanolamine deaminase from Salmonella typhimurium has been determined by using isotope labeling and techniques of electron paramagnetic resonance (EPR) spectroscopy. The presence of nitrogen is detected from the difference in the EPR line shapes of the product radicals that are cryotrapped during steady-state turnover on either 14N- or 15N-labeled aminoethanol substrate. Three-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy of the product radical labeled with 2H reveals two types of beta-2H hyperfine couplings. A structural model is proposed in which the two beta-2H couplings arise from two C1-C2 product radical rotamer states. The sum of the dihedral angles between the C2 p-orbital axis and C1-Hbeta bonds is 120 degrees , which indicates sp3-hybridization at C1. This confirms the C1 carbinolamine structure. The identification of the carbinolamine product radical indicates that the radical rearrangement in ethanolamine deaminase deviates from the solution elimination reaction pathway and proceeds by migration of the amine from C2 of the substrate radical to C1 of the product radical.

Spin–spin interaction in ethanolamine deaminase

The adenosylcobalamin coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium catalyzes the deamination of aminoethanol to acetaldehyde and ammonia. The radical intermediate observed during steady state turnover of substrate aminoethanol has been characterized by continuous wave electron paramagnetic resonance (EPR) spectroscopy [J. Am. Chem. Soc. 121 (1999) 10522]. This study presents simulations of EPR spectra of this radical intermediate. Quantitative fits to the EPR spectra are achieved with a model of isotropic exchange and magnetic dipolar interaction between the substrate-derived radical and the Co II in the corrin ring. The simulated parameters are compared with those of substrate analog 2-aminopropanol-derived radical in the same enzyme. The comparison confirms that the aminoethanol-derived product radical interacts more weakly with the Co II than the 2-aminopropanol-derived radical and suggests that the reduction of isotropic exchange between the aminoethanol-derived product radical and the Co II is probably due to orientational-dependent wave function overlap. Successful fits to the radical line shapes of different isotope substitutions unequivocally establish that the observed radical intermediate is an π-electron-based product radical. The derived principal hyperfine values for the 13C α and 1H α nucleus are consistent with previous electron nuclear double resonance (ENDOR) studies on similar radicals, thus providing reliable experimental hyperfine coupling constants for comparison with quantum mechanical-based calculations to gain further insight into the molecular structure of the observed radical.

Geometry of Reactant Centers in the CoII-Substrate Radical Pair State of Coenzyme B12-Dependent Ethanolamine Deaminase Determined by Using Orientation-Selection-ESEEM Spectroscopy

The distances and orientations among the C5‘ methyl group of 5‘-deoxyadenosine, the radical-bearing C1 carbon of the substrate radical, and the low spin (S=1/2) CoII in cob(II)alamin in the active site of coenzyme B12-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized in the CoII-substrate radical pair state by using two-pulse X-band electron spin−echo electron paramagnetic resonance (ESE-EPR) and electron spin−echo envelope modulation (ESEEM) spectroscopies in the disordered solid state. Our approach is based on the orientation-selection created in the EPR spectrum of the biradical by the axial electron−electron dipolar interaction. Simulation of the ESE-EPR line shape yielded CoII-radical exchange and dipole interaction terms, which were used to calculate the CoII-C1 distance of 11.1 A and the dependence of the EPR line shape on the angle between the CoII-C1 axis and the magnetic field vector. ESEEM spectroscopy performed at four magnetic field values addressed the coup...

Interaction of the substrate radical and the 5'-deoxyadenosine-5'-methyl group in vitamin B(12) coenzyme-dependent ethanolamine deaminase.

The distance and relative orientation of the C5' methyl group of 5'-deoxyadenosine and the substrate radical in vitamin B(12) coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized by using X-band two-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy in the disordered solid state. The (S)-2-aminopropanol-generated substrate radical catalytic intermediate was prepared by cryotrapping steady-state mixtures of enzyme in which catalytically exchangeable hydrogen sites in the active site had been labeled by previous turnover on (2)H(4)-ethanolamine. Simulation of the time- and frequency-domain ESEEM requires two types of coupled (2)H. The strongly coupled (2)H has an effective dipole distance (r(eff)) of 2.2 A, and isotropic coupling constant (A(iso)) of -0.35 MHz. The weakly coupled (2)H has r(eff) = 3.8 A and A(iso) = 0 MHz. The best (2)H ESEEM time- and frequency-domain simulations are achieved with a model in which the hyperfine couplings arise from one strongly coupled hydrogen site and two equivalent weakly coupled hydrogen sites located on the C5' methyl group of 5'-deoxyadenosine. This model indicates that the unpaired electron on C1 of the substrate radical and C5' are separated by 3.2 A and are thus at closest contact. The close proximity of C1 and C5' indicates that C5' of the 5'-deoxyadenosyl moiety directly mediates radical migration between cobalt in cobalamin and the substrate/product site over a distance of 5-7 A in the active site of ethanolamine deaminase.

Identification of a Rearranged-Substrate, Product Radical Intermediate and the Contribution of a Product Radical Trap in Vitamin B12 Coenzyme-Dependent Ethanolamine Deaminase Catalysis

Identification of a Rearranged-Substrate, Product Radical Intermediate and the Contribution of a Product Radical Trap in Vitamin B₁₂ Coenzyme-Dependent Ethanolamine Deaminase Catalysis

Identification of dimethylbenzimidazole axial coordination and characterization of (14)N superhyperfine and nuclear quadrupole coupling in Cob(II)alamin bound to ethanolamine deaminase in a catalytically-engaged substrate radical-Cobalt(II) biradical state.

Cobalt(II)-(14)N superhyperfine and (14)N nuclear quadrupole couplings in cryotrapped free and ethanolamine deaminase-bound cob(II)alamin have been characterized in the disordered solid state by using X-band electron spin-echo envelope modulation (ESEEM) spectroscopy. Enzyme-bound cob(II)alamin was cryotrapped after formation by substrate-initiated, thermally activated cleavage of the cobalt-carbon bond of adenosylcobalamin. Free dimethylbenzimidazole axial base-on cob(II)alamin was formed by photolysis of the corresponding adenosylcobalamin and cryotrapped in glycerol-aqueous glass. Three-pulse ESEEM experiments were performed by using microwave pulse excitation at the g( perpendicular) value of Co(II) at magnetic field values of 287.0 and 345.0 mT and over a range of tau values from 227 to 1316 ns. Two common sets of (14)N features are distinguished in the ESEEM spectra. One set is assigned to the remote (N1) nitrogen in the dimethylbenzimidazole alpha-axial ligand by using two independent approaches: (a) comparison of ESEEM from cob(II)alamin with ESEEM from cob(II)inamide-ligand model compounds and (b) from the correspondence between the N1 (14)N nuclear quadrupole parameters derived from ESEEM simulations and those computed by using density functional theory. The second set is assigned to the corrin ring (14)N nuclei. The results identify the coenzyme's on-board dimethylbenzimidazole moiety as the alpha-axial ligand to cob(II)alamin in ethanolamine deaminase in the substrate radical-Co(II) biradical catalytic intermediate state. Thus, Co(II) is a pentacoordinate, alpha-axial liganded complex during turnover. We infer that dimethylbenzimidazole is also the alpha-axial ligand to the intact coenzyme in the resting enzyme. A 14% increase in the isotropic hyperfine coupling of the remote dimethylbenzimidazole (14)N nucleus in enzyme-bound versus free base-on cob(II)alamin shows an enhanced delocalization of unpaired spin density from Co(II) onto the axial ligand, which would contribute to the acceleration of the cobalt-carbon bond cleavage rate in situ.

Interactions of Substrate and Product Radicals with CoII in Cobalamin and with the Active Site in Ethanolamine Deaminase, Characterized by ESE-EPR and 14N ESEEM Spectroscopies

Interactions of the cryotrapped 2-aminopropanol-1-yl substrate radical and aminoethanol-derived product radical catalytic intermediates with the active site of vitamin B12 coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium in disordered frozen aqeuous solution have been characterized by using X-band electron spin−echo electron paramagnetic resonance (ESE-EPR) and electron spin−echo envelope modulation (ESEEM) spectroscopies performed at 6 K. ESE-EPR spectra show that the doublet splitting of the radical line shape by electron−electron exchange and dipolar interactions with CoII in cob(II)alamin is stronger for the substrate radical (11.1 mT) than for the product radical (7.1 mT). The aminoethanol-derived product radical unpaired spin density at C2 is therefore positioned closer to CoII than the 2-aminopropanol-1-yl substrate radical unpaired spin density at C1. Multifrequency three-pulse ESEEM spectra obtained at g = 2.00 for each radical display the same τ- and magnetic field-dependent...

Evidence for the B12-dependent enzyme ethanolamine deaminase in Salmonella.

THE enteric bacteria Escherichia coli and Salmonella typhimurium, like man, do not synthesise significant amounts of cobalamin (B12) compounds and thus depend on exogenous vitamin B12 for their B12-dependent enzymes1. In E. coli and S. typhimurium the only reported B12-dependent enzyme catalyses the final step in methionine biosynthesis—the methylation of homocysteine to give methionine. These bacteria do not depend on exogenous B12 for growth, however, for they have an alternative B12-independent homocysteine transmethylase (non-B12 enzyme) which can catalyse the same reaction. The non-B12 transmethylase is much less efficient than the corresponding B12-dependent enzyme: in bacteria grown in the absence of vitamin B12, the non-B12-dependent enzyme comprises 3–5 % of the total soluble protein2. If this enzyme is blocked by mutation, the mutant bacteria require either methionine itself or exogenous B12, which allows them to utilise the B12-dependent enzyme for methionine biosynthesis3. We have sought other B12-dependent enzymes in S. typhimurium and found evidence for an ethanolamine deaminase. We did not find other examples of parallel non-B12-dependent and B12-dependent enzymes in S. typhimurium. E. coli also seems to have an ethanolamine deaminase.

Microbial metabolism of amino alcohols. Aminoacetone metabolism via 1-aminopropan-2-ol in Pseudomonas sp. N.C.I.B. 8858.

1. Pseudomonas sp. N.C.I.B. 8858 grew well on d- and l-1-aminopropan-2-ol and on aminoacetone. 2. Cell-free extracts possessed high activities of inducibly formed l-1-aminopropan-2-ol-NAD(+) oxidoreductase, amino alcohol-ATP phosphotransferase, dl-1-aminopropan-2-ol O-phosphate phospho-lyase and aldehyde-NAD(+) oxidoreductase, but no 1-aminopropan-2-ol racemase or d-1-aminopropan-2-ol-NAD(+) oxidoreductase. 3. The amino alcohol kinase (activated by ADP) was non-stereospecific towards 1-aminopropan-2-ol and was one-third as active with ethanolamine. The phospho-lyase was active with l- and d-1-aminopropan-2-ol O-phosphate, but ethanolamine O-phosphate was only one-tenth as active as its higher homologues. The purified aldehyde dehydrogenase was active with propionaldehyde, acetaldehyde and also with methylglyoxal. The previously observed 2-oxo aldehyde dehydrogenase activity was considered to be due to the broadly specific aldehyde dehydrogenase. 4. Mutants of Pseudomonas sp. N.C.I.B. 8858 deficient in 1-aminopropan-2-ol kinase, 1-aminopropan-2-ol O-phosphate phospho-lyase, aldehyde dehydrogenase or an enzyme involved in propionate metabolism were incapable of growth on aminoacetone or 1-aminopropan-2-ol as carbon source, although all except the kinase- or phospho-lyasedeficient mutants could use these compounds and ethanolamine as nitrogen sources. The aldehyde dehydrogenase-deficient mutants produced copious amounts of propionaldehyde and acetaldehyde during growth on the corresponding amino alcohols. 5. The path of aminoacetone metabolism in Pseudomonas sp. N.C.I.B. 8858 was concluded to involve l-1-aminopropan-2-ol, the O-phosphate ester of this compound, propionaldehyde and propionate as obligatory intermediates. d-1-Aminopropan-2-ol was metabolized by the same route as the l-isomer, gratuitously inducing formation of the stereospecific l-1-aminopropan-2-ol dehydrogenase. 6. Extracts of the pseudomonad grown with ethanolamine as the nitrogen source were devoid of 1-aminopropan-2-ol dehydrogenase, the kinase and the phospho-lyase, but exhibited cobamide coenzyme-dependent deaminase activity. Mutants deficient in kinase or phospho-lyase (deaminating) grew well on ethanolamine as the nitrogen source. Ethanolamine deaminase was inactive with, but inhibited by, 1-aminopropan-2-ol.

Nicotinic Acid Metabolism: VII. MECHANISMS OF ACTION OF CLOSTRIDIAL α-METHYLENEGLUTARATE MUTASE (B12-DEPENDENT) AND METHYLITACONATE ISOMERASE

Abstract Methylitaconate (MeIT) is identified as the intermediate in the conversion of α-methyleneglutarate (αMG) to dimethylamaleate (DMM). α-Methyleneglutarate mutase catalyzes the B12 coenzyme-dependent conversion of α-methyleneglutarate to methylitaconate. Analogous to other B12-dependent reactions the coenzyme serves in this process as a carrier of the hydrogen that is abstracted from αMG and replaced by the acrylyl moiety of αMG through an intramolecular rearrangement. That carbon 5' of the 5'-deoxyadenosyl moiety covalently bonded to the cobalt is the specific site on the B12 coenzyme involved in the hydrogen carrying function was shown by (a) incorporation of tritium from [5'-3H]dimethylbenzimidazolycobamide coenzyme (DMBC) into both the substrate and the product of αMG mutase and (b) transfer of tritium from 2-amino[1-3H]-ethanol to MeIT and αMG in a coupled system containing αMG mutase, unlabeled B12 coenzyme, and ethanolamine deaminase, an enzyme known to transfer tritium to the 5'-methylene group of the coenzyme from 2-amino[1-3H]ethanol. Interaction of labeled B12 coenzyme with the two purified enzymes in the coupled reaction and proportionality of tritium recovery in products to amount of [5'-3H]DMBC added indicates a dissociable enzyme-coenzyme complex or a transfer of tritium between enzyme-bound and free coenzyme. The tritium transferred from [3H]DMBC to αMG is incorporated into one (or both) of the methylene carbons of the 5-carbon chain of the molecule and none is present in the α-methylene group. The solvent, water, does not appear to participate in the hydrogen transfer reaction catalyzed by αMG mutase. The second isomerization involving the migration of the double bond of methylitaconate to form dimethylmaleate is catalyzed by a sulfhydryl enzyme, methylitaconate isomerase. Using 3H2O and 2H2O, proton exchange with the solvent during this reaction was demonstrated. A hydrogen on position 3 of MeIT and in the reverse direction on a methyl group of DMM exchanges with the solvent.

The Mechanism of Action of Ethanolamine Deaminase: VI. ETHYLENE GLYCOL, A QUASI-SUBSTRATE FOR ETHANOLAMINE DEAMINASE

Abstract Incubation of 5'-deoxyadenosylcobalamin with ethylene glycol in the presence of ethanolamine deaminase leads to the cleavage of the coenzyme at the carbon-cobalt bond, with the production of equivalent amounts of 5'-deoxyadenosine and acetaldehyde, together with a new corrinoid which appears to arise by a substitution in the coordination sphere of the cobalt whereby the 5'-deoxyadenosyl residue is replaced by another ligand. This reaction is accompanied by the transfer of a hydrogen from ethylene glycol to 5'-deoxyadenosine. After the reaction is over the acetaldehyde dissociates slowly from the enzyme, but both 5'-deoxyadenosine and the new corrinoid remain bound to the enzyme, where the latter is gradually converted to hydroxocobalamin. The cleavage of coenzyme, which is first order in enzyme-B12 complex, obeys saturation kinetics with respect to ethylene glycol, showing a Vmax of about 0.2 sec-1 and a Km for ethylene glycol of 0.02 m. The optical spectrum of the new B12 derivative resembles that of an alkyl cobalamin or a thiol cobalamin. However, spectral changes observed on treating the reaction mixture with urea or heat to denature the enzyme suggest that the latter possibility is more likely than the former. The possibility that the new compound is a hitherto unknown type of corrin derivative cannot be excluded. As a result of the reaction, the activity of the enzyme is substantially reduced, but not abolished. The enzyme is capable of promoting the cleavage of more than 1 mole of coenzyme per mole of active site. Both of these observations indicate that the enzyme participates catalytically in the cleavage reaction, and is not destroyed as a consequence of the reaction. These results are formulated in terms of a mechanism which relates the observed reaction to the catalytic reaction in which ethanolamine is substrate.

The Mechanism of Action of Ethanolamine Deaminase: III. Inhibition by coenzyme B12 analogues

Abstract A study was carried out on the effect of a large number of B12 analogues on the coenzyme B12-dependent enzyme ethanolamine deaminase. The analogues tested included MeB12, EtB12, AB12, RB12, BB12, IB12, DB12, UB12, CNB12, and OHB12. DB12 was found to be capable of replacing the coenzyme. In contrast, all the rest of the analogues were inhibitors. With all the inhibitors except IB12, the inhibition was progressive, with the reaction velocity decaying exponentially with time to a final, constant value. The extent of inhibition by IB12 was independent of time; kinetics showed that this inhibition was competitive with respect to the coenzyme. Inhibition by MeB12 was also found to be competitive with DMBC, when its effect of the initial reaction rate was considered. Studies of the time-dependent increase in the extent of inhibition observed with MeB12 showed that the rate constant for this process (the deactivation constant) followed saturation kinetics with respect to the concentration of inhibitor. A similar dependence of the deactivation constant on the inhibitor concentration was observed with RB12. The time-dependent decrease in the reaction rate caused by this derivative was found to be irreversible. Kinetic studies performed at the end of the decay period showed that RB12 behaved as a competitive inhibitor with respect to the coenzyme, but that its Ki at the end of this period was much lower than the Ki estimated for the beginning of the reaction. Furthermore, the Vm for ethanolamine deaminase at the end of the decay period was only one-sixth of the Vm of the uninhibited enzyme. Light scattering studies and studies of the effect of enzyme concentration on the characteristics of the decay indicated that the inhibition is not associated with polymerization or depolymerization of the enzyme. Experiments with tritiated MeB12 showed that the progressive inhibition induced by this derivative is not accompanied by cleavage of the carbon-cobalt bond. Titration experiments with RB12 have shown that the progressive inhibition is fully expressed at an inhibitor to enzyme ratio of 2:1. These results can be explained by a model in which the presence of inhibitor induces a change in the enzyme from the form isolated in the purification to a form with a lower Vm and a much greater affinity for the inhibitor. The abbreviations used are: MeB12, methylcobalamin; EtB12, β-hydroxyethylcobalamin; AB12, β-(2-tetrahydropyryloxy)ethylcobalamin; RB12, 1-O-methyl-5-deoxyribosylcobalamin; BB12, δ-(9-adenyl)butylcobalamin; IB12, 5'-deoxyinosylcobalamin; DB12, 2',5'-dideoxyadenosylcobalamin; UB12, 5'-deoxyuridylcobalamin; CNB12, cyanocobalamin; OHB12, hydroxocobalamin; DMBC, 5'-deoxyadenosylcobalamin.

The Mechanism of Action of Ethanolamine Deaminase: IV. Cobamide-dependent binding of substrate to ethanolamine deaminase

Abstract In the presence of a number of alkyl cobalamins, a complex is formed between ethanolamine deaminase and radioactive ethanolamine which can be isolated by gel filtration. Studies with the methylcobalamin-dependent complex have shown that the amount of substrate bound reaches a maximum at a cobamide to enzyme ratio of 2. The binding of substrate to this complex is depressed by ethanolamine analogues which are competitive inhibitors of the catalytic reaction. Measurements of binding as a function of substrate concentration indicate a dissociation constant for the complex of 2 x 10-7 m, a value which is surprisingly high in view of the apparent stability of the complex. Incubating the complex with unlabeled ethanolamine or allowing it to reside on the gel column for various periods of time leads to a loss of radioactivity from the complex which is almost complete by 6 min. This loss does not occur if the complex is exposed to light prior to incubation with unlabeled substrate. However, exposure of a mixture of enzyme and methylcobalamin to light in the absence of substrate prevents the complex from forming when radioactive substrate is added. Although the photolyzed complex is stable in the presence of unlabeled substrate, treatment with 6 m urea results in the release of all the radioactivity from the complex. With both the unphotolyzed and the photolyzed complexes, the radioactive compound associated with the enzyme was shown to be unchanged ethanolamine. These results are interpreted as suggesting that the formation of the enzyme-substrate complex is reversible in practical terms, but that in the presence of certain cobalamin derivatives the dissociation of the complex is slowed to the extent that it can be observed experimentally. Thus, the apparent stability of the complex is kinetic rather than thermodynamic in origin.

The mechanism of action of ethanolamine deaminase. V. The photolysis of enzyme-bound alkylcobalamins.

The mechanism of action of ethanolamine deaminase. V. The photolysis of enzyme-bound alkylcobalamins.

The mechanism of action of ethanolamine deaminase, II. The spectrum of the ethanolamine deaminase-coenzyme B 12 complex during the act of catalysis

The mechanism of action of ethanolamine deaminase, II. The spectrum of the ethanolamine deaminase-coenzyme B 12 complex during the act of catalysis

An EPR signal generated by the ethanolamine deaminase-coenzyme B 12 complex in the presence of substrate.

Abstract EPR spectroscopy of a mixture of ethanolamine deaminase, 5′-deoxy-adenosylcobalamin and deuterated ethanolamine frozen during the act of catalysis revealed the appearance of a signal at g = 2.09. This signal disappeared when the reaction was permitted to continue until the substrate was exhausted, but reappeared upon subsequent addition of substrate. No signal was observed when enzyme or coenzyme was omitted from the reaction mixture, but small signals did appear when substrate was omitted. There were significant differences between these latter signals and the signal obtained from a sample containing all three components. This evidence is consistent with the hypothesis that species with unpaired electrons are involved in the mechanism of action of ethanolamine deaminase.