Enzymatic radical catalysis: coenzyme B12-dependent diol dehydratase

A peculiar function resides in a peculiar structure. Coenzyme B₁₂ or adenosylcobalamin, a naturally occurring organometallic compound, serves as a cofactor for enzymatic radical reactions. How do the enzymes form catalytic radicals at the active sites? How do the enzymes utilize and control the high reactivity of the radicals for catalysis? Recently, three‐dimensional structures of several radical‐containing or radical‐forming enzymes including B₁₂ enzymes have been reported, enabling the analysis of the fine mechanisms of the action of these interesting enzymes. Our biochemical, mutational, and crystallographic studies as well as theoretical calculations on diol dehydratase, an adenosylcobalamin–dependent enzyme, revealed that its structure is adapted for its function—that is, activation of the Co? C bond toward homolysis, abstraction of a specific hydrogen atom from the substrate and its recombination to a particular product, and transition state stabilization in the hydroxyl group migration of a substrate‐derived radical. The functions of K⁺ and the active‐site amino acid residues in enzyme catalysis are also investigated. Based on the results, the fine mechanism of the enzyme and the energetic feasibility of enzymatic radical catalysis are described here. © 2002 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 2: 352–366, 2002: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.10035     

Acid-, base-, and lewis-acid-catalyzed heterolysis of methoxide from an alpha-hydroxy-beta-methoxy radical: models for reactions catalyzed by coenzyme B12-dependent diol dehydratase

[Reaction: see text].A model for glycol radicals was employed in laser flash photolysis kinetic studies of catalysis of the fragmentation of a methoxy group adjacent to an alpha-hydroxy radical center. Photolysis of a phenylselenylmethylcyclopropane precursor gave a cyclopropylcarbinyl radical that rapidly ring opened to the target alpha-hydroxy-beta-methoxy radical (3). Heterolysis of the methoxy group in 3 gave an enolyl radical (4a) or an enol ether radical cation (4b), depending upon pH. Radicals 4 contain a 2,2-diphenylcyclopropane reporter group, and they rapidly opened to give UV-observable diphenylalkyl radicals as the final products. No heterolysis was observed for radical 3 under neutral conditions. In basic aqueous acetonitrile solutions, specific base catalysis of the heterolysis was observed; the pK(a) of radical 3 was determined to be 12.5 from kinetic titration plots, and the ketyl radical formed by deprotonation of 3 eliminated methoxide with a rate constant of 5 x 10(7) s(-1). In the presence of carboxylic acids in acetonitrile solutions, radical 3 eliminated methanol in a general acid-catalyzed reaction, and rate constants for protonation of the methoxy group in 3 by several acids were measured. Radical 3 also reacted by fragmentation of methoxide in Lewis-acid-catalyzed heterolysis reactions; ZnBr2, Sc(OTf)3, and BF3 were found to be efficient catalysts. Catalytic rate constants for the heterolysis reactions were in the range of 3 x 10(4) to 2 x 10(6) s(-1). The Lewis-acid-catalyzed heterolysis reactions are fast enough for kinetic competence in coenzyme B12 dependent enzyme-catalyzed reactions of glycols, and Lewis-acid-catalyzed cleavages of beta-ethers in radicals might be applied in synthetic reactions.     

Stabilisation of methylene radicals by cob(II)alamin in coenzyme B12 dependent mutases

Coenzyme B12 initiates radical chemistry in two types of enzymatic reactions, the irreversible eliminases (e.g., diol dehydratases) and the reversible mutases (e.g., methylmalonyl-CoA mutase). Whereas eliminases that use radical generators other than coenzyme B12 are known, no alternative coenzyme B12 independent mutases have been detected for substrates in which a methyl group is reversibly converted to a methylene radical. We predict that such mutases do not exist. However, coenzyme B12 independent pathways have been detected that circumvent the need for glutamate, beta-lysine or methylmalonyl-CoA mutases by proceeding via different intermediates. In humans the methylcitrate cycle, which is ostensibly an alternative to the coenzyme B12 dependent methylmalonyl-CoA pathway for propionate oxidation, is not used because it would interfere with the Krebs cycle and thereby compromise the high-energy requirement of the nervous system. In the diol dehydratases the 5'-deoxyadenosyl radical generated by homolysis of the carbon-cobalt bond of coenzyme B12 moves about 10 A away from the cobalt atom in cob(II)alamin. The substrate and product radicals are generated at a similar distance from cob(II)alamin, which acts solely as spectator of the catalysis. In glutamate and methylmalonyl-CoA mutases the 5'-deoxyadenosyl radical remains within 3-4 A of the cobalt atom, with the substrate and product radicals approximately 3 A further away. It is suggested that cob(II)alamin acts as a conductor by stabilising both the 5'-deoxyadenosyl radical and the product-related methylene radicals.     

Computational exploration of rearrangements related to the vitamin B12-dependent ethanolamine ammonia lyase catalyzed transformation

DFT (B3LYP/6-31G) and ab initio molecular orbital theory (QCISD/cc-pVDZ) are used to investigate several possible mechanisms involving free radical intermediates as well as their protonated forms for processes related to the coenzyme B(12)-dependent rearrangement catalyzed by ethanolamine ammonia lyase. Two major types of rearrangements are discussed in detail, intramolecular migration and dissociation of the amine/ammonia groups, for both of which several scenarios are considered. According to the calculations, the complete dissociation of the migrating group and its subsequent association constitute an unlikely route for both the protonated and the unprotonated reactant because of the high-energy barriers (more than 23 kcal/mol) involved in these steps. Direct migration of the protonated amine group is far more favorable (10.4 kcal/mol) and therefore presents the most likely candidate for the actual enzymatic reaction. The calculations further imply that the direct loss of an ammonium cation (10.6 kcal/mol) represents a feasible pathway as well. Comparing the rearrangements for the aminoethanol radical and its protonated counterpart, in line with previous findings reported by Golding, Radom, and co-workers, we find that the migration of a protonated group is in general associated with lower energy barriers, suggesting that the actual enzyme substrate quite likely corresponds to (partially) protonated aminoethanol. As the extent of the substrate protonation/deprotonation by the active site of the enzyme may vary, the actual energy barriers are expected to range between the values calculated for the two extreme cases of a substrate, that is, the aminoethanol radical 2 and its fully protonated form 6.     

Homocoenzyme B12 and bishomocoenzyme B12: covalent structural mimics for homolyzed, enzyme-bound coenzyme B12

Efficient electrochemical syntheses of "homocoenzyme B(12)" (2, Co(beta)-(5'-deoxy-5'-adenosyl-methyl)-cob(III)alamin) and "bishomocoenzyme B(12)" (3, Co(beta)-[2-(5'-deoxy-5'-adenosyl)-ethyl]-cob(III)alamin) are reported here. These syntheses have provided crystalline samples of 2 and 3 in 94 and 77 % yield, respectively. In addition, in-depth investigations of the structures of 2 and 3 in solution were carried out and a high-resolution crystal structure of 2 was obtained. The two homologues of coenzyme B(12) (2 and 3) are suggested to function as covalent structural mimics of the hypothetical enzyme-bound "activated" (that is, "stretched" or even homolytically cleaved) states of the B(12) cofactor. From crude molecular models, the crucial distances from the corrin-bound cobalt center to the C5' atom of the (homo)adenosine moieties in 2 and 3 were estimated to be about 3.0 and 4.4 A, respectively. These values are roughly the same as those found in the two "activated" forms of coenzyme B(12) in the crystal structure of glutamate mutase. Indeed, in the crystal structure of 2, the cobalt center was observed to be at a distance of 2.99 A from the C5' atom of the homoadenosine moiety and the latter was found to be present in the unusual syn conformation. In solution, the organometallic moieties of 2 and 3 were shown to be rather flexible and to be considerably more dynamic than the equivalent group in coenzyme B(12). The homoadenosine moiety of 2 was indicated to occur in both the syn and the anti conformations.     

Catalytic roles of the metal ion in the substrate-binding site of coenzyme B12-dependent diol dehydratase

Functions of the metal ion in the substrate-binding site of diol dehydratase are studied on the basis of quantum mechanical/molecular mechanical (QM/MM) calculations. The metal ion directly coordinates to substrate and is essential for structural retention and substrate binding. The metal ion has been originally assigned to the K(+) ion; however, QM/MM computations indicate that Ca(2+) ion is more reasonable as the metal ion because calculated Ca-O distances better fit to the coordination distances in X-ray crystal structures rather than calculated K-O distances. The activation energy for the OH group migration, which is essential in the conversion of diols to corresponding aldehydes, is sensitive to the identity of the metal ion. For example, the spectator OH group of substrate is fully deprotonated by Glu170 in the transition state for the OH group migration in the Ca-contained QM/MM model, and therefore the barrier height is significantly decreased in the model having Ca(2+) ion. On the other hand, the deprotonation of the spectator OH group cannot effectively be triggered by the K(+) ion. Moreover, in the hydrogen recombination, the most energy-demanding step is more favorable in the Ca-contained model. The proposal that the Ca(2+) ion should be involved in the substrate-binding site is consistent with an observed large deuterium kinetic isotope effect of 10, which indicates that C-H bond activation is involved in the rate-determining step. Asp335 is found to have a strong anticatalytic effect on the OH group migration despite its important role in substrate binding. The synergistic interplay of the O-C bond cleavage by Ca(2+) ion and the deprotonation of the spectator OH group by Glu170 is required to overcome the anticatalytic effect of Asp335.     

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.     

Ligand substitution behavior of a simple model for coenzyme B12

The ligand substitution reactions of trans-[CoIII(en)2(Me)H2O]2+, a simple model for coenzyme B12, were studied for cyanide and imidazole as entering nucleophiles. It was found that these nucleophiles displace the coordinated water molecule trans to the methyl group and form the six-coordinate complex trans-[Co(en)2(Me)L]. The complex-formation constants for cyanide and imidazole were found to be (8.3 +/- 0.7) x 10(4) and 24.5 +/- 2.2 M-1 at 10 and 12 degrees C, respectively. The second-order rate constants for the substitution of water were found to be (3.3 +/- 0.1) x 10(3) and 198 +/- 13 M-1 s-1 at 25 degrees C for cyanide and imidazole, respectively. From temperature and pressure dependence studies, the activation parameters delta H++, delta S++, and delta V++ for the reaction of trans-[CoIII(en)2(Me)H2O]2+ with cyanide were found to be 50 +/- 4 kJ mol-1, 0 +/- 16 J K-1 mol-1, and +7.0 +/- 0.6 cm3 mol-1, respectively, compared to 53 +/- 2 kJ mol-1, -22 +/- 7 J K-1 mol-1, and +4.7 +/- 0.1 cm3 mol-1 for the reaction with imidazole. On the basis of reported activation volumes, these reactions follow a dissociative mechanism in which the entering nucleophile could be weakly bound in the transition state.     

Computational mutation analysis of hydrogen abstraction and radical rearrangement steps in the catalysis of coenzyme B12-dependent diol dehydratase

A mutation analysis of the catalytic functions of active-site residues of coenzyme B(12)-dependent diol dehydratase in the conversion of 1,2-propanediol to 1,1-propanediol has been carried out by using QM/MM computations. Mutants His143Ala, Glu170Gln, Glu170Ala, and Glu170Ala/Glu221Ala were considered to estimate the impact of the mutations of His143 and Glu170. In the His143Ala mutant the activation energy for OH migration increased to 16.4 from 11.5 kcal mol(-1) in the wild-type enzyme. The highest activation energy, 19.6 kcal mol(-1), was measured for hydrogen back-abstraction in this reaction. The transition state for OH migration is not sufficiently stabilized by the hydrogen-bonding interaction formed between the spectator OH group and Gln170 in the Glu170Gln mutant, which demonstrates that a strong proton acceptor is required to promote OH migration. In the Glu170Ala mutant, a new strong hydrogen bond is formed between the spectator OH group and Glu221. A computed activation energy of 13.6 kcal mol(-1) for OH migration in the Glu170Ala mutant is only 2.1 kcal mol(-1) higher than the corresponding barrier in the wild-type enzyme. Despite the low activation barrier, the Glu170Ala mutant is inactive because the subsequent hydrogen back-abstraction is energetically demanding in this mutant. OH migration is not feasible in the Glu170Ala/Glu221Ala mutant because the activation barrier for OH migration is greatly increased by the loss of COO(-) groups near the spectator OH group. This result indicates that the effect of partial deprotonation of the spectator OH group is the most important factor in reducing the activation barrier for OH migration in the conversion of 1,2-propanediol to 1,1-propanediol catalyzed by diol dehydratase.     

电化学及光谱电化学方法研究辅酶B~12 模型化合物的生成与解离.I.

本文用电化学及光谱电化学方法研究了钴卟啉与苄基氯的相互作用,表明形成有σ钴-碳键的辅梅B~12模型化合物的前提条件是钴卟啉的中心钴氧化态必须降低到I价.讨论了σ钴-碳键的形成和解离与电位的关系,现场检测到辅酶B~12模型化合物的形成和解离的紫外可见光谱,提出了钴 中啉与苄基氯的ECF作用机理.     

New developments in the field of vitamin B12: enzymatic reactions dependent upon corrins and coenzyme B12.

Simple corrins such as vitamin B₁₂ and vitamin B₁₂ coenzyme catalyze a variety of unusual enzymatic reactions of which some are still without analogy in organic or organometallic chemistry. The mechanisms of these reactions are currently the subject of lively discussion. The present review focuses attention on new ideas about the mode of action of vitamin B₁₂ coenzymes in enzymatic reactions.     

Purification of the coenzyme B12-containing 2-methyleneglutarate mutase from Clostridium barkeri by high-performance liquid chromatography.

Two methods are described by which the enzymes 2-methyleneglutarate mutase and 3-methylitaconate delta-isomerase from Clostridium barkeri have been separated by high-performance liquid chromatography on a much larger scale than reported previously. First, the mutase eluted before the delta-isomerase after incubation with the mild detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS) followed by high-performance anion-exchange chromatography on Mono Q in the presence of the same detergent. Second, an even better separation, although with a lower yield of mutase, was obtained by hydrophobic interaction chromatography on phenyl-Sepharose HiLoad, whereby the enzymes were eluted in the reverse order. Final high-performance anion-exchange chromatography of the latter preparation on Mono Q at pH 8 gave highly purified 2-methyleneglutarate mutase (greater than 95% purity) which had a pink-orange colour (lambda max 280, 375, 470 and 532 nm). The enzyme was active in the absence of coenzyme B12 (adenosylcobalamin) and contained 2.1 mol of this coenzyme per homotetramer (molecular mass, m = 300 kilodalton).     

Rotation of the exo-methylene group of (R)-3-methylitaconate catalyzed by coenzyme B(12)-dependent 2-methyleneglutarate mutase from Eubacterium barkeri

2-Methyleneglutarate mutase from the anaerobe Eubacterium (Clostridium) barkeri is an adenosylcobalamin (coenzyme B(12))-dependent enzyme that catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Two possibilities for the mechanism of the carbon skeleton rearrangement of the substrate-derived radical to the product-related radical are considered. In both mechanisms an acrylate group migrates from C-3 of 2-methyleneglutarate to C-4. In the "addition-elimination" mechanism this 1,2-shift occurs via an intermediate, a 1-methylenecyclopropane-1,2-dicarboxylate radical, in which the migrating acrylate is simultaneously attached to both C-3 and C-4. In the "fragmentation-recombination" mechanism the migrating group, a 2-acrylyl radical, becomes detached from C-3 before it starts bonding to C-4. In an attempt to distinguish between these two possibilities we have investigated the action of 2-methyleneglutarate mutase on the stereospecifically deuterated substrates (Z)-3-methyl[2'-(2)H(1)]itaconate and (Z)-3-[2'-(2)H(1),methyl-(2)H(3)]methylitaconate. The enzyme catalyzes the equilibration of both compounds with their corresponding E-isomers and with a 1:1 mixture of the corresponding (E)- and (Z)-2-methylene[2'-(2)H(1)]glutarates, as shown by monitoring of the reactions with (1)H and (2)H NMR. In the initial phase of the enzyme-catalyzed equilibration a significant excess (8-11%) of (E)-3-methyl[2'-(2)H(1)]itaconate over its equilibrium value was observed ("E-overshoot"). The E-overshoot was only 3-4% with (Z)-3-[2'-(2)H(1),methyl-(2)H(3)]methylitaconate because the presence of the deuterated methyl group raises the energy barrier from 3-methylitaconate to the corresponding radical. The overshoot is explained by postulating that the migrating acrylate group has to overcome an additional energy barrier from the state leading back to the substrate-derived radical to the state leading forward to the product-related radical. It is concluded that the fragmentation-recombination mechanism can provide an explanation for the results in terms of an additional energy barrier, despite the higher calculated activation energy for this pathway.