Dioldehydrase Reaction Research Articles

Identification of cis-ethanesemidione as the organic radical derived from glycolaldehyde in the suicide inactivation of dioldehydrase and of ethanolamine ammonia-lyase.

The hydrate of glycolaldehyde is a substrate analogue that induces the formation of cob(II)alamin and 5'-deoxyadenosine from adenosylcobalamin at the active site of dioldehydrase, and the resulting complex is inactive. The carbon atoms of glycolaldehyde hydrate remain bound to this complex, and it has been postulated that the first step or steps of the catalytic process on glycolaldehyde hydrate generate an intermediate that undergoes a destructive side reaction leading to inactivation of the enzyme [Wagner, O. W., Lee, H. A., Jr., Frey, P. A., and Abeles, R. H. (1966) J. Biol. Chem. 249, 1751-1762]. All evidence suggests that dioldehydrase reaction proceeds by a radical mechanism, and the glycolaldehyde hydrate is expected to be converted initially into a radical. Electron paramagnetic resonance (EPR) spectroscopic analysis of the inactivated complex shows that glycolaldehyde is transformed into a cis-ethanesemidione radical that is weakly spin-coupled to the cob(II)alamin in the active site of the enzyme. This radical has been identified by analysis of EPR spectra obtained from samples with (13)C- and (2)H-labeled forms of glycolaldehyde. The analysis shows that the stable radical associated with the inactive complex is symmetrical and that it contains a single solvent-exchangeable proton, consistent with a cis-ethanesemidione. Glycolaldehyde also inactivates ethanolamine ammonia-lyase (EAL). EPR studies of ethanolamine ammonia-lyase reveal that treatment with glycolaldehyde also results in formation of an ethanesemidione radical bound in the active site. The suicide inactivation in both enzymatic reactions is postulated to result from formation of this stable radical, which cannot react further to abstract a hydrogen atom from 5'-deoxyadenosine. Analysis of the electron spin-spin coupling between the semidione radicals and cob(II)alamin in both enzymes indicates that the distance between the radical and Co(2+) is approximately 11 A in each case.

The Dehydration Step in the Enzyme-Coenzyme-B12 Catalyzed Diol Dehydrase Reaction of 1,2-Dihydroxyethane Utilizing a Hydrogen-Bonded Carboxylic Acid Group as an Additional Cofactor: A Computational Study

The various steps in a mechanism for the diol dehydrase reaction in which a carboxylic acid group of an amino acid residue at the active site of the enzyme serves as an additional cofactor have been investigated using density functional theory (B3LYP) calculations. This mechanism involves a neutral radical rather than a protonated radical (radical cation). 1,2-Dihydroxyethane was chosen as the substrate, and formic acid was selected as a model for the carboxylic acid group. The 1,2-dihydroxyeth-l-yl radical (produced by H-atom transfer from the substrate to the 5‘-deoxyadenosyl radical) forms a nine-membered ring structure with the formic acid. There are two intermolecular hydrogen bonds in this ring structureone with the OH of the ĊOH group in the radical as a hydrogen donor and the CO group in the formic acid as an acceptor, and the other with the OH of the COH group in the radical as an acceptor and the COH group of the formic acid as a hydrogen donor. Bond rearrangement within this hydrogen-bonded ring structure results in the formation of a hydrogen-bonded product in which transfer of the radical center from one carbon atom to the adjacent carbon atom has taken place. Fission of the CO bond at the new radical center leads to the elimination of H2O and the separation of the formylmethyl radical from which acetaldehyde is formed by H-atom transfer. The interchange of the HOC and CO bonding in the carboxylic acid group is an overall feature of the mechanism. Furthermore, like the radical, the substrate diol is found to form a nine-membered ring structure with formic acid; this contains two intermolecular hydrogen bonds analogous to those formed between the radical and formic acid. This finding provides support for the hypothesis that two-point attachment of the substrate via its HO groups to the enzyme occurs prior to the H-atom transfer, which initiates the dehydration process. Geometries, energies, and entropies are reported for the hydrogen-bonded reactant ring structure, for the transition state, for a hydrogen-bonded product structure, and for all the separate molecules. Enthalpy, entropy, and free energy changes for the various steps have been calculated from these data, which relate to the gas phase, and for the corresponding reactions at the active site where the restricted spatial environment results in a much diminished translational entropy. Modified entropy values have accordingly been employed by taking the liquid state as the model, evaluating using the empirical equation, = + 15.8. Further calculations suggest that polarization within the protein cavity containing the active site has a very small effect on the barrier height and the exothermicity of the dehydration process. Rate constants calculated from computed free energies of activation for the dehydration via an HO-bridge structure (transition state), and via fragmentation giving the HO-radical and syn-vinyl alcohol, are far smaller than the experimentally determined rate constant, whereas that calculated for the formic acid cofactor mechanism is of the same order of magnitude as the experimental value.

The Function of the Nucleotide Loop of Adenosylcobalamin in Diol Dehydrase Reaction

The Function of the Nucleotide Loop of Adenosylcobalamin in Diol Dehydrase Reaction

Importance of the Nucleotide Loop Moiety Corrdinated to the Cobalt Atom of Adenosylcobalamin for Coenzymic Function in the Diol Dehydrase Reaction.

Three analogs of adenosylcobalamin were synthesized and their coenzymic properties in the diol dehydrase reaction were studied. Neither adenosylcobinamide nor adenosylcobinamide phosphate was active as coenzyme and showed very low affinity for apoenzyme, irrespective of the presence of nucleotide loop fragments, such as 5,6-dimethylbenzimidazole, alpha-D-ribazole, or alpha-D-ribazole-3'-phosphate. The coordination of pyridine to the cobalt atom neither confers the coenzymic function upon adenosylcobinamide nor strengthens the inhibitory effect of cyanoaquacobinamide and methylcobinamide significantly. The analog of adenosylcobalamin in which the N-3 position of 5,6-dimethylbenz-imidazole is methylated was also not active as coenzyme and showed very low affinity for apoenzyme. Since 3,5,6-trimethylbenzimidazole in this analog is no longer coordinated to the cobalt atom, these results show that at least a part of the nucleotide loop moiety coordinated to the cobalt atom of adenosylcobalamin is essential for tight binding to the apoenzyme and therefore for manifestation of coenzymic function.

Roles of the D-ribose and 5,6-dimethylbenzimidazole moieties of the nucleotide loop of adenosylcobalamin in manifestation of coenzymic function in the diol dehydrase reaction.

Coenzyme analogs in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analog containing imidazole instead of 5,6-dimethylbenzimidazole were synthesized. Coordination of the 5,6-dimethylbenzimidazole to the cobalt atom in these analogs was much weaker than that in cobalamins. The replacement of this base with imidazole did not significantly alter the strength of the coordination to the cobalt atom. 5,6-Dimethylbenzimidazolyl trimethylene and tetramethylene and imidazolyl trimethylene analogs were partially active as coenzymes in the diol dehydrase reaction in this order as judged by kcat, but the others were not active as coenzymes and were weak competitive inhibitors. This indicates that neither the alpha-D-ribofuranose ring nor the functional groups of the ribose moiety are essential for coenzymic function. There was an optimum loop size of the analogs for catalysis and for tight binding to the apoenzyme, which corresponds to the loop size of cobalamins. Therefore, the D-ribose moiety seems important as a spacer to keep the base in the proper position. The reaction with the imidazolyl trimethylene analog as coenzyme was accompanied with concomitant rapid inactivation during catalysis. The inactivation occurred only in the presence of substrate. Upon inactivation with this analog, 5'-deoxyadenosine and a B12r-like species were formed from the adenosyl group and the rest of the analog molecule, respectively, without modification of the apoenzyme. Therefore, it can be concluded that this is a kind of suicide inactivation which occurred from one of the intermediates in the normal catalytic process. The dimethylbenzo moiety of the regular coenzyme thus seems to play an important role in preventing the intermediate complexes from inactivation during catalysis.

Studies on the mechanism of the adenosylcobalamin-dependent diol dehydrase reaction by the use of analogs of the coenzyme.

A series of 16 analogs of 5'-deoxy-5'-adenosylcobalamin (adenosylcobalamin) were examined for their effects on the diol dehydrase system of Klebsiella pneumoniae (Aerobacter Aerogenes). Four analogs, ara-adenosyl-, aristeromycyl-, 3-isoadenosyl-, and nebularylcobalamin, were able to function as coenzymes in the diol dehydrase reaction, coenzyme activity decreasing in that order. Like the native holoenzyme, complexes of the enzyme with these four analogs show a cob(II)alamin-like absorption peak or shoulder in the presence of 1,2-propanediol. Analogs containing hypoxanthine, cytosine, or benzimidazole do not function as coenzymes, but are weak competitive inhibitors in the presence of adenosylcobalamin. Analogs in which the D-ribosyl moiety is replaced by L-ribose or by an alkyl chain of 2 to 6 carbons are inactive as coenzymes, but act as competitive inhibitors with extremely high affinity for the apoenzyme. Complexes with the inactive analogs showed visible spectra similar to those of the corresponding free cobalamins. Upon anaerobic photolysis and subsequent aeration, complexes with the first group of inactive analogs produced unusually stabilized cob(II)alamin, while complexes with the second group of inactive analogs were readily photolyzed to a hydroxocobalamin-enzyme complex. Complexes with adeninylpentyl- and L-adenosylcobalamin were stable to light under the same conditions. These findings suggest that both the ribose and the adenine moiety of the nucleoside participate in enzyme-coenzyme interaction, involving not only the binding to the apoenzyme but also the activation of the carbon-cobalt bond.

Preparation, properties and biological activities of succinyl derivatives of vitamin B 12

Three new derivatives of vitamin B 12, O 2′-succinyl-, O 5′-succinyl-, and O 2′. O 5′—disuccinyl-vitamin B 12, whose α-ribose moieties of the nucleotide ligand are succinylated, were prepared by reaction of the vitamin with succinic anhydride. The first succinylation took place rapidly and almost predominantly on 5′-OH of α-ribose, and the second succinylation much more slowly on 2′-OH. From the behaviors in paper electrophoresis and the lability to CN − of cobalt-base bond of O 2′-succinylated vitamin B 12 derivatives, it was suggested that the terminal COOH of the O 2′-succinyl group forms an inner salt with the imidazole nucleus of 5,6-dimethylbenzimidazole. Monosuccinyl- and disuccinyl-vitamin B 12 were easily reconverted into vitamin B 12 by mild acid or base hydrolysis. Heating at 130° for 5 min also led to the complete severance of the succinyl group of O 5′-succinyl-vitamin B 12. None of the three succinyl derivatives inhibited the diol dehydrase reaction when added with coenzyme B 12, suggesting that the ability to bind to the apoenzyme is strongly diminished or almost lost by succinylation on 2′- or 5′-OH of α-ribose. None of the succinyl vitamin B 12 compounds showed either biological activity or anti-vitamin B 12 activity when tested with Escherichia coli 215, a methionine-B 12 auxotroph. None of them significantly inhibited [ 3H] vitamin B 12 uptake by E. coli 215 cells. This observation implies that succinyl derivatives of vitamin B 12 are hardly incorporated into the cells of E. coli.

Relative enantiomer binding and reaction rates with propanediol dehydrase

In the dioldehydrase reaction, L(+) propanediol reacts at a faster rate than the D(−)-isomer in competitive reactions, but when the diols are run independently the rate of the D(−)-isomer is 2.3 times greater than that of the L(+)-isomer. It is assumed that this reversal is due to a difference in the Michaelis-Menten constants. A ratio of K mD(−)/K mL(+) = 3.2 ± 0.3 at 25° is calculated from a series of competitive reactions. It is concluded that the binding site is such that while both isomers are accomodated readily, the D(−)-isomer is hindered to some degree in its approach to the binding site. However, once the enzyme-substrate complex is formed, the D(−)-isomer is in a more favorable configuration for further reaction.

Studies on the mechanism of hydrogen transfer in the coenzyme B12 dependent dioldehydrase reaction II.

Studies on the mechanism of hydrogen transfer in the coenzyme B12 dependent dioldehydrase reaction II.

Cobamides and Ribonucleotide Reduction: VI. Enzyme-Catalyzed Hydrogen Exchange Between Water and Deoxyadenosylcobalamin

Abstract When deoxyadenosylcobalamin was incubated with ribonucleoside triphosphate reductase from Lactobacillus leichmannii, dihydrolipoate, and a nucleoside triphosphate in the presence of tritiated water, labeled hydrogen was incorporated into nonexchangeable positions in the cobamide. A maximum of 1.4 atoms of labeled hydrogen was incorporated per molecule of deoxyadenosylcobalamin. On photolysis of the labeled cobamide the radioactivity was found in the 5'-hydrogen atoms of the nucleoside products derived from the deoxyadenosyl moiety. This result indicates that the hydrogen atoms of the methylene group bound to cobalt in the cobamide exchange with water, a conclusion confirmed by nonenzymic synthesis of 5-deoxyadenosylcobalamin-5'-3H2 and the demonstration that tritium is transferred from this compound into water. This hydrogen exchange occurred only in the presence of enzyme, dithiol, and a nucleotide. The dithiol requirement could be met by 1,3- or 1,4-dithiols or by the thioredoxin system, but monothiols were relatively ineffective. Ribonucleoside triphosphates could satisfy the nucleotide requirement, but deoxyribonucleoside triphosphates were also active and dGTP was the most effective nucleotide of all. Nucleoside mono- and diphosphates were less effective than the corresponding triphosphates. The specificity of the nucleotide requirement, the absence of hydrogen transfer to or from the activating nucleotide, and the fact that deoxynucleotides did not undergo detectable reaction in the system suggest that nucleotides act as allosteric activators of the hydrogen exchange reaction. The enzymic transfer of hydrogen between water and the 5'-methylene group of the cobamide is considered to occur via the dithiol and to indicate the reversible formation of a reduced cobamide intermediate, probably identical with that occurring in the mutase reactions that require deoxyadenosylcobalamin and in the diol dehydrase reaction. In ribonucleotide reduction this reduced cobamide intermediate is regarded as the immediate donor of a hydride ion to position 2' α of the nucleotide substrate. Attempts to obtain evidence of the existence of this intermediate by incorporating label from deoxyadenosylcobalamin-5'-3H2 into the deoxynucleotide product of the reductase or by dilution by unlabeled deoxyadenosylcobalamin of tritium incorporation from water into deoxynucleotide product were unsuccessful. However, as required by the hypothesis, inactivation of the enzyme for the hydrogen transfer reaction under various conditions always resulted in parallel or greater inactivation for ribonucleotide reduction.

Studies on the Mechanism of Hydrogen Transfer in the Cobamide Coenzyme-dependent Dioldehydrase Reaction

Abstract When dl-1,2-propanediol-1-3H is converted to propionaldehyde in the presence of dioldehydrase and cobamide coenzyme, tritium is transferred to the coenzyme. The tritiated coenzyme so obtained transfers tritium to the reaction product when reacted with dl-1,2-propanediol and apoenzyme. The coenzyme is tritiated exclusively at the C-5' position of the adenosyl moiety. The location of tritium was established by chemical degradation and further confirmed by showing that chemically synthesized cobamide coenzyme, containing tritium at the C-5' position, transferred tritium to the product when added to enzyme and unlabeled substrate. The conversion of dl-1,2-propanediol-1-3H to propionaldehyde proceeds with inter- and intramolecular tritium transfer. Approximately 1% of the tritiated substrate reacts by intramolecular transfer, i.e. the hydrogen abstracted from C-1 of a substrate molecule is found in the α position of the aldehyde derived from that molecule. A partial reaction occurs, as evidenced by tritium exchange between tritiated coenzyme and propionaldehyde or acetaldehyde under conditions in which no net reaction occurs. The results obtained have led to the following tentative reaction sequence. Hydrogen is abstracted from C-1 of dl-1,2-propanediol and transferred to the coenzyme, where it becomes equivalent with at least one, but probably both, hydrogens of the C-5' position. This results in the formation of a reduced form of the coenzyme and a molecule derived through the oxidation of the substrate. In a subsequent step the hydrated form of propionaldehyde is formed by a transfer of hydrogen from the reduced coenzyme to the intermediate derived from the substrate.

The Mechanism of Action of Cobamide Coenzyme in the Ribonucleotide Reductase Reaction

When highly purified cobamide-dependent ribonucleotide reductase from Lactobacillus leichmannii is incubated with synthetically prepared 5, 6-dimethylbenzimidazolylcobamide 5'-deoxyadenosyl coenzyme containing tritium attached to carbon atom 5' of the deoxyadenosyl moiety (DBCC-5'-3H), tritium is transferred from DBCC-5'-3H to H2O in a reaction requiring substrate, enzyme, and dithiol reductant. At low enzyme concentrations, the amount of tritium transferred to H2O is stoichiometrically equivalent to the amount of ribonucleotide reduced; hence, substrate-dependent release of tritium from DBCC-5'-3H is a sensitive assay for cobamide-dependent ribonucleotide reductase. When enzyme is incubated with unlabeled cobamide coenzyme in H2O-3H, tritium is transferred to coenzyme as well as to the 2'-deoxyribosyl carbon of the reaction product. The amount of tritium transferred from H2O-3H to product decreases with increasing concentration of unlabeled coenzyme. Coenzyme tritiated in the reductase reaction labels propionaldehyde in the dioldehydrase reaction. The results indicate that cobamide coenzyme functions as an essential hydrogen-transferring agent in the cobamide-dependent ribonucleotide reductase reaction, and that transferred hydrogen attaches to carbon atom 5' of the coenzyme deoxyadenosyl moiety. We conclude that although hydrogen is transferred intermolecularly in the reductase reaction and intramolecularly in the dioldehydrase reaction, the mechanism of coenzyme function is the same in both reactions.