Diol Dehydrase Research Articles

The binding site for the adenosyl group of coenzyme B 12 in diol dehydrase

The binding of cob(II)alamin (Cbl II) and 5′-deoxyadenosine to diol dehydrase was studied spectroscopically and with [U- 14C]5′-deoxyadenosine. Cbl II was bound to this enzyme forming a tight 1:1 complex which was resistant to oxidation by O 2 even in the presence of CN −. An irreversible 1:1:1 ternary complex was formed between enzyme, Cbl II, and 5′-deoxyadenosine, when the enzyme was incubated first with the nucleoside and then with Cbl II. When this order of addition of the constituents was reversed, no 5′-deoxyadenosine was bound to the enzyme-Cbl II complex. Hydroxocobalamin could also bind to the enzyme together with the nucleoside, while other cob(III)alamins bearing a bulkier Coβ ligand displaced the nucleoside upon binding to the enzyme. The binding of [U- 14C]5′-deoxyadenosine was strongly inhibited by unlabeled 5′-deoxy- ara-adenosine, 4′,5′-anhydroadenosine, adenosine, adenine, and 5′,8-cyclic adenosine, in this order, but not by 5′-deoxyuridine. These results constitute direct evidence for the presence of the binding site for the adenosyl group of adenosylcobalamin, which is spatially limited to and highly specific for adenine nucleosides. The binding of 5′-deoxyadenosine to the apoenzyme was reversible.

Ligand exchange reactions of diol dehydrase-bound cobalamins and the effect of the nucleoside binding.

The inactive complex of diol dehydrase with hydroxocobalamin was resolved by treatment with SO2-3, followed by dialysis to remove SO2-3, giving the apoenzyme which was reconstitutable into catalytically active holoenzyme upon addition of adenosylcobalamin ("re-activation"). Spectral evidence showed that the enzyme-bound hydroxocobalamin undergoes a Co beta-ligand exchange reaction forming sulfitocobalamin. Sulfitocobalamin was bound to diol dehydrase only loosely, and therefore dissociated from the enzyme. In contrast, neither the enzyme-hydroxocobalamin-5'-deoxyadenosine nor the enzyme-hydroxocobalamin-adenosine complex was resolved and thus re-activated by this procedure. It was shown spectroscopically that the hydroxocobalamin in these complexes does not react with SO2-3, or even with CN-, indicating that the OH group in the Co beta-position was blocked spatially by these enzyme-bound nucleosides. Neither O2-inactivated holoenzyme nor the holoenzyme inactivated suicidally by glycerol or 1,2-ethanediol during dehydration reaction was also re-activated by the same procedure. The complex of the enzyme with cyanocobalamin or methylcobalamin was not resolvable by the SO2-3 treatment. This was because these cobalamins bound to the enzyme were not subject to a ligand exchange reaction with SO2-3.

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Vitamin B12-dependent enzymes catalyze various methyl transfer reactions and isomerizations accompanied by the initial 1, 2-shift rearrangement. Adenosylcobalamin, which involves both metal-coordinate and organometallic bonds in the same molecule, acts as a true catalyst in the latter reactions. Coenzyme reaction machanisms have been extensively investigated particularly in connection With methylmalonyl-CoA mutase and diol dehydrase, and homolytic cleavage of the Co-C bond as well as its heterolytic cleavage to yield a carbanion intermediate has been postulated. Since cob (I) alamin and its model compounds in Co (I) state act as supernucleophiles, various reactions as catalyzed by these complexes have been examined. For the establishment of effective B12 holoenzyme models, synthetic lipids which form stable single-walled bilayer aggregates in aqueous media are plausibly adopted as apoenzyme models. Novel organometallic chemistry is hopefully developed in the light of biomimetic notions.

Chemical modification of coenzyme B 12-dependent diol dehydrase with pyridoxal 5′-phosphate: Lysyl residue essential for interaction between two components of the enzyme

The apoenzyme of diol dehydrase was inactivated by modification with pyridoxal 5′-phosphate (pyridoxal- P). The inactivation was accompanied by appearance of a new peak at 425 nm which was shifted to 325 nm by reduction with NaBH 4. ϵ- N-Pyridoxyl lysine was detected by paper chromatography and paper electrophoresis from the hydrolysate of the NaBH 4-reduced enzyme-pyridoxal- P complex. The relationship of inactivation vs pyridoxal- P incorporation as well as kinetic experiments suggests that one lysyl residue per enzyme molecule was essential for catalytic activity, although two to three pyridoxal- P molecules were introduced into the almost completely inactivated enzyme molecule. Both 1,2-propanediol (substrate) and adenosylcobalamin (coenzyme) completely protected the enzyme from inactivation. The result of disc gel electrophoresis showed that the inactivation of diol dehydrase by pyridoxal- P results from irreversible dissociation of the enzyme into subunits upon pyridoxal- P modification. Therefore, it is suggested that this modifiable lysyl residue is essential for subunit interaction to form an active oligomeric enzyme. The inactivated enzyme restored activity by addition of excess component F, but not by S, suggesting that the essential lysyl residue is located in component F of the enzyme. Pyridoxal- P-modified enzyme was no longer able to bind cyanocobalamin (a competitive inhibitor of adenosylcobalamin).

Reactive sulfhydryl groups of coenzyme B 12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neither p-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.

Coenzyme B 12-dependent diol dehydrase: Chemical modification with 2,3-butanedione and phenylglyoxal

The apoenzyme of diol dehydrase was inactivated by two arginine-specific reagents, 2,3-butanedione and phenylglyoxal, in borate buffer. In both cases, the inactivation followed pseudo-first-order kinetics. Kinetic data show that the incorporation of a single reagent molecule per active site of the enzyme is necessary for the complete inactivation. The modification with 2,3-butanedione was reversed by dilution of the reagent and borate concentrations (65% activity recovered). 1,2-Propanediol (substrate) partially protected the enzyme against inactivation. The holoenzyme was almost insensitive to 2,3-butanedione and phenylglyoxal, indicating that the essential arginine residue is prevented from the attack of these reagents either by direct blockage with the bound coenzyme or by an indirect conformational change caused by coenzyme binding. The inactivation of diol dehydrase by 2,3-butanedione did not result in dissociation of the enzyme into subunits. From these results, we concluded that the essential arginine residue is located at or in close proximity to the active site of diol dehydrase.

The synthesis of several immobilized derivatives of vitamin B12 coenzyme and their use as affinity adsorbents for a study of interactions of diol dehydrase with the coenzyme.

The synthesis of several immobilized derivatives of vitamin B12 coenzyme and their use as affinity adsorbents for a study of interactions of diol dehydrase with the coenzyme.

Model experiments relevant to the mechanism of adenosylcobalamin-dependent diol dehydrase: further investigations of isomerisations of dihydroxyalkyl radicals

Anaerobic photolysis of 4,5-dihydroxypentyl(pyridine)cobaloxime in 0.1M acetic acid (pH 3) gives ca. 10% of pentanal, whereas 1,1,5,5-tetradeuterio-4,5-dihydroxypentyl(pyridine)cobaloxime affords 0.6% of 1,5,5,5-tetradeuteriopentanal. Under similar conditions, 5-deuterio-5,6-dihydroxyhexyl(pyridine)cobaloxime gives ca. 1% of 6-deuteriohexan-2-one and 4% of 2-deuteriohexanal, in contrast to 5,6-dihydroxyhexyl(pyridine)cobaloxime which affords 16% of hexan-2-one and 4% of hexanal. These results support a mechanism for the photocon-version of dihydroxyalkylcobaloximes into aldehydes or ketones at pH 3, in which light-induced homolysis of the Co–C σ-bond to give a dihydroxyalkyl radical is followed by a 1,5-hydrogen shift. The resulting isomeric dihydroxyalkyl radical, possessing a hydroxy-group at the radical centre, undergoes an acid-catalysed transformation to aldehyde or ketone. With the deuteriated cobaloximes the 1,5-H shift is impeded by a primary isotope effect (kH/kDca. 20). Anaerobic photolysis of 4,5-dihydroxycyclo-octyl(pyridine)cobaloxime at pH 3 yields ca. 40% of cyclo-octanone. This reaction owes its efficiency to a favourable transannular 1,5-H shift which converts the 4,5-dihydroxycyclo-octyl radical into the 1,2-dihydroxycyclo-octyl radical. The relevance of the above reactions to adenosylcobalamin-dependent reactions catalysed by diol dehydrase is discussed.

8] Immobilized derivatives of vitamin B12 coenzyme and its analogs

Publisher Summary This chapter describes immobilized derivatives of vitamin B12 coenzyme and its analogs. The immobilized derivatives of cobalamins are useful not only for purification of cobalamin-dependent enzymes or cobalamin-binding proteins, but also for basic studies on biospecific interactions between cobalamins and apoenzymes or cobalamin binders. Vitamin B12 coenzyme (adenosylcobalamin) is an organocobalt compound that has a very complicated structure. The molecule is composed of the three major parts; a corrin ring (equatorial quadridentate), a lower nucleotide ligand moiety (axial fifth ligand), and an upper nucleoside ligand moiety. Immobilization of corrinoids is accomplished by either (a) reaction of ω-aminoalkyl-Sepharose with the carboxylic acid derivatives of corrinoids in the presence of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide; or (b) reaction of cyanogen bromide-activated Sepharose with the amino derivatives of Corrinoids. This chapter describes methods for preparing several new corrinoid affinity adsorbents and also briefly describes their application for a basic study of the interaction of the cobalamin coenzyme with the apoenzyme of cobalamin-dependent diol dehydrase.

Bacterial metabolism of propane-1,2-diol

The pathway of propane-1,2-diol metabolism by a species of Flavobacterium able to grow on the diol as the sole source of carbon was influenced by the degree of aeration of the growth medium. Under strongly aerobic conditions the diol was exclusively catabolised to lactaldehyde by an initial diol oxidase, subsequently metabolised to pyruvate and then oxidised to CO 2 by the tricarboxylic acid cyle. Under microaerophilic conditions some propane-1,2-diol was catabolised by the oxidase-initiated pathway, but some diol was alternatively catabolised by an inducible diol dehydrase to propionaldehyde and subsequently reduced to n-propanol as an end product of metabolism.

Coenzyme B 12-dependent diol dehydrase: Purification, subunit heterogeneity, and reversible association

A purification procedure for diol dehydrase ( dl-1,2-propanediol hydro-lyase, EC 4.2.1.28) of Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 has been developed which gives the highest specific activity for this enzyme obtained so far. The purified enzyme is homogeneous by the criteria of ultracentrifugation ( s 20,w = 8.9 S) and disc gel electrophoresis in the presence of substrate. The molecular weight of approximately 230,000 was obtained by gel filtration and ultracentrifugal sedimentation equilibrium. The enzyme is composed of components F and S whose molecular weights were determined to be approximately 26,000 and 200,000, respectively, by gel filtration. The incubation of both components F and S with the substrate leads to complete reassociation of the components. Disc gel electrophoresis in the presence of sodium dodecyl sulfate and terminal amino acid analyses indicate that component S consists of at least four nonidentical subunits. The reversible association and heterogeneity of the subunits were also demonstrated with the crude enzyme by immunoelectrophoresis.

A model system based on photodecompositions of alkylcobaloximes for the conversion of 1,2-diols to aldehydes catalysed by diol dehydrase

Attempts to model the conversion of 1,2-diols to aldehydes catalysed by the adenosylcobalamin-dependent enzyme diol dehydrase utilising the photohomolysis of alkylcobaloximes, have been carried out in two ways. First, the alkyl radical released from such photolysis has been shown to convert ethane-1,2-diol to CH3CHO at pH 2 with an efficiency of ca. 10%. Second, photolysis of several compounds of the type HOCH2CHOH[CH2]n-Co(dmgH)2(C5H5N) yields products (when n= 3 or 4) suggestive of the sequence: (a) photohomolysis followed by (b) an intramolecular 1,5-hydrogen shift (n= 3 or 4) or 1,6-hydrogen shift (n= 4) to produce a 1,2-dihydroxyalkyl radical which in turn undergoes (c) further transformations, including either an acid-catalysed 1,2-hydroxy shift [to give a 1-(dihydroxymethyl)alkyl radical], or acid-catalysed dehydration to R1ĊHCOR2(R1= Prn, R2= H, R1= H, R2= Bun, or R1= Bun, R2= H), finally yielding R1CH2COR2. When n= 3, the exclusive carbonylcontaining product from the dihydroxyalkyl group is pentanal (via a 1,5-hydrogen shift). These results enable us to counter claims that radical chemistry cannot simulate the diol dehydrase reactions, and to provide a critique of the present discussion of models for adenosylcobalamin-dependent enzymatic reactions.

Mechanism of action of adenosylcobalamin: glycerol and other substrate analogs as substrates and inactivators for propanediol dehydratase - kinetics, stereospecificity, and mechanism

A number of vicinal diols were found to react with propanediol dehydratase, typically resulting in the conversion of enzyme-bound adenosylcobalamin to cob(II)alamin and formation of aldehyde or ketone derives from substrate. Moreover, all are capable of effecting the irreversible inactivation of the enzyme. The kinetics and mechanism of product formation and inactivation were investigated. Glycerol, found to be a very good substrate for diol dehydratase as well as a potent inactivator, atypically, did not induce cob(II)alamin formation to any detectable extent. With glycerol, the inactivation process was accompanied by conversion of enzyme-bound adenosylcobalamin to an alkyl or thiol cobalamin, probably by substitution of an amino acid chain near the active site for the 5'-deoxy-5'-adenosyl ligand on the cobalamin. The inactivation reaction with glycerol as the inactivator exhibits a deuterium isotope effect of 14, strongly implicating hydrogen transfer as an important step in the mechanism of inactivation. The isotope effect on the rate of product formation was found to be 8.0. Experiments with isotopically substituted glycerols indicate that diol dehydrase distinguishes between "R" and "S" binding conformations, the enzyme-(R)-glycerol complex being predominately responsible for the product-forming reaction, while the enzyme-(S)-glycerol complex results primarily in the activation reaction. Mechanistic implications are discussed. A method for removing enzyme-bound hydroxycobalamin that is nondestructive to the enzyme and a technique for measuring the binding constants of (R)- and (S)-1,2-propanediols are presented.

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.

Substrate specificity of coenzyme B 12-dependent diol dehydrase: Glycerol as both a good substrate and a potent inactivator

The substrate specificity of adenosylcobalamin-dependent diol dehydrase was further studied in detail using an enzyme preparation that appears homogeneous by ultracentrifugal and gel electrophoretical criteria. Besides 1,2-propanediol and 1,2-ethanediol, glycerol, 1,2- and 2,3-butanediol were found to serve as substrate for the enzyme, whereas 1,3-propanediol was not. Of the substrate analogs tested, glycerol displayed some striking features: it was dehydrated to β-hydroxypropionaldehyde with concomitant inactivation of the enzyme. Although the initial velocity with glycerol was comparable to that with 1,2-propanediol, the dehydration reaction ceased almost completely within 3 min accompanying rapid, irreversible inactivation of the holoenzyme. 1,2- and 2,3-Butanediol were converted to butyraldehyde and methyl ethyl ketone, respectively, at a rate much lower than that with 1,2-propanediol. 2,3-Butanediol is the only compound, other than 1,2-diols, known at present to show a considerable substrate activity.

Mechanism of action of adenosylcobalamin: 3-fluoro-1,2-propranediol as substrate for propanediol dehydrase--mechanistic implications.

3-Fluro-1,2-propanediol has been found to be a substrate for propanediol dehydrase and has very similar binding and catalytic constants compared to the natural substrate. The only isolable products of the reaction are acrolein and inorganic fluoride; with 3-fluoro-3,3-dideuterio-1,2-propanediol as substrate, only 3,3-dideuterioacrolein is obtained. These results indicate that the primary product of the reaction is 3-fluoropropionaldehyde which spontaneously loses hydrogen fluoride to yield acrolein. The similar kinetic parameters for the fluorinated as compared to the normal substrate suggest that significant charge does not develop on the fluorinated or, by implication, the natural substrate during any rate-limiting steps of the reaction. These results support a radical, as contrasted to an ionic pathway for reactions involving adenosylcobalamin and diol dehydrase.

Immobilized diol dehydrase and its use in studies of cobalamin binding and subunit interaction.

Coenzyme B12 dependent diol dehydrase from Aerobacter aerogenes was immobilized by covalent binding to CNBr-activated Sepharose 4B. The Sepharose-bound enzyme exhibited a markedly high catalytic activity, viz., 75-95% of the specific activity of the original free enzyme. The apoenzyme acquired much greater stability to heat by immobilization. No significant difference between the immobilized and free enzymes was observed in the following properties: the affinity for coenzyme B12; the sensitivity to a sulfhydryl-modifying agent; the absolute requirement for a certain monovalent cation, such as K+, for catalysis; the susceptibility toward oxygen upon incubation with coenzyme B12 in the absence of substrate. These results suggest that the structure and function of the enzyme are not significantly influenced by immobilization on Sepharose. The immobilized enzyme was found to provide a convenient method for a study of ligand interaction with the enzyme. The subunit interaction between two dissimilar subunits, components F and S, was investigated using the component S immobilized on CNBr-activited Sepharose and free component F, and it was demonstrated that the substrate (1,2-propanedoil) promotes the hybrid formation between component F and component S, but K+ alone rather retarded the subunit association to some extent. Na+ markedly weakens the forces which bind the subunits together. The relationship between cobalamin binding and subunit structure is also discussed.

Coenzyme action of adenosyl-13-epicobalamin in the diol dehydrase system

Coenzyme action of adenosyl-13-epicobalamin in the diol dehydrase system

A physical explanation of the EPR spectrum observed during catalysis by enzymes utilizing coenzyme B

We have proposed that the “doublet” EPR spectra observed during catalysis by a number of coenzyme B 1,2-requiring enzymes arises from a weak electrostatic exchange interaction between an organic free radical and low spin Co(II), B 1,2r. By varying the magnitude of the exchange coupling we have quite accurately simulateed the published EPR spectra from the enzyme systems: diol dehydrase, glycerol dehydrase, ribonucleotide reductase, and ethanolamine ammonia lyase. A dipolar model was shown to be incompatible with the observed properties of these systems.

Additions and Corrections - Diol Dehydrase Model Studies. The Acid Catalyzed Rearrangement of β-Hydroxyisopropylcobaloxime

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAdditions and Corrections - Diol Dehydrase Model Studies. The Acid Catalyzed Rearrangement of β-HydroxyisopropylcobaloximeKenneth Brown and Lloyd IngrahamCite this: J. Am. Chem. Soc. 1975, 97, 14, 4152Publication Date (Print):July 1, 1975Publication History Published online4 February 2004Published inissue 1 July 1975https://doi.org/10.1021/ja00847a607RIGHTS & PERMISSIONSArticle Views18Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (125 KB) Get e-Alerts Get e-Alerts