Study on the mechanism of action of adenosylcobalamin-dependent glycerol dehydratase from Aerobacter aerogenes

Yakusheva, M. I.; Poznanskaya, A. A.; Поспелова, T. А.; Рудакова, И. П.; Yurkevich, A. M.; Yakovlev, V.A.

doi:10.1016/0005-2744(77)90127-9

Cited by 27 publications

(12 citation statements)

References 29 publications

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“…Thermolysis of AdoCbi + with RS - / RSH Present. Because a cysteinethiolate has been suggested in the literature as a conceivable axial ligand for enzyme-bound AdoCbl, we briefly examined the products and kinetics of AdoCbi + thermolysis at 100 °C in phosphate-buffered, pH 7.2 H 2 O with the dithiol dithioerythritol (DTE; 25 °C, p K a 1 = 9.2, p K a 2 = 9.9;40a 100 °C, p K a 1 = ca. 6.8, p K a 2 = ca.…”

Section: Resultsmentioning

confidence: 99%

Adocobinamide, the Base-off Analog of Coenzyme B₁₂ (Adocobalamin). 2.¹ Probing the “Base-on” Effect in Coenzyme B₁₂ via Cobalt−Carbon Bond Thermolysis Product and Kinetic Studies as a Function of Exogenous Pyridine Bases

1996

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The thermolysis of the Co-C bond in adocobinamide (AdoCbi + BF 4 -) in anaerobic ethylene glycol has been studied as a function of a series of para-substituted pyridine axial bases using the TEMPO radical-trapping method. In contrast to the slower rates of Co-C cleavage previously found for benzylcobinamide, neopentylcobinamide, and the (R-phenylethyl)cobaloxime coenzyme B 12 models, for AdoCbi + the rate of total Co-C cleavage becomes faster as the para-substituted pyridines become more electron-donating. Specifically, the 110 °C k obsd for AdoCbi + BF 4 -total Co-C cleavage increased 23-fold on going from 1 M pyridine (py) to 1 M p-(dimethylamino)pyridine (Me 2 N-py). However, HPLC product studies reveal that the percentage of abiological Co-C heterolysis increases (to a limiting value); that is, Co-C heterolysis is a major reason for the observed rate increase seen for Me 2 N-py. Deconvolution of the k obsd rate constant into its heterolysis and homolysis components yields values of the 110 °C k heterolysis and k homolysis for AdoCbi + ‚base for Me 2 N-py and pyridine. These data in turn reveal that the AdoCbi + BF 4 -base-on homolysis rate constant does not increase within experimental error as one goes from py to the more basic Me 2 N-py (k on,h ) 8(3) × 10 -4 and 7(1) × 10 -4 s -1 , respectively), but that the base-on heterolysis rate constant changes by 17-fold (k on,het ) 0.4(0.1) × 10 -4 and 7(1) × 10 -4 s -1 for py and Me 2 N-py, respectively). The plausible biological significance of these results is then discussed, notably the heretofore unsubstantiated idea, first suggested by Mealli, Sabat, and Marzilli, that one major evolutionary pressure for selecting and appending the 5,6-dimethylbenzimidazole axial base in coenzyme B 12 is because this base limits Co-C bond heterolysis, thereby promoting the biologically relevant Co-C cleavage reaction, Co-C homolysis.

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Section: Resultsmentioning

confidence: 99%

Adocobinamide, the Base-off Analog of Coenzyme B₁₂ (Adocobalamin). 2.¹ Probing the “Base-on” Effect in Coenzyme B₁₂ via Cobalt−Carbon Bond Thermolysis Product and Kinetic Studies as a Function of Exogenous Pyridine Bases

1996

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“…Thus, ground-state distortion of the already large (121−124° ) Co−C−C bond angle in AdoCbl could represent an effective mechanism for catalyzing Co−C bond cleavage. Studies of the enzymatic activity of analogues of AdoCbl altered in the structure of the Ado ligand, some of which are partially active coenzymes and some of which are inactive inhibitors, − provide some experimental support for this hypothesis. The AdoCbl analogue N 1 -deaza-AdoCbl is 56% as active as AdoCbl and N 3 -deaza-AdoCbl is 46% as active as AdoCbl with diol dehydratase, while N 10 -monomethyl-AdoCbl has 12% of the activity of AdoCbl with the glycerol dehydratase of Aerobacter aerogenes .…”

Section: Discussionmentioning

confidence: 99%

Activation Parameters for the Carbon−Cobalt Bond Homolysis of Coenzyme B₁₂Induced by the B₁₂-Dependent Ribonucleotide Reductase fromLactobacillus leichmannii

Brown

1998

J. Am. Chem. Soc.

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The temperature dependence of the kinetics of the rapid, reversible cleavage of the carbon−cobalt bond of 5‘-deoxyadenosylcobalamin (AdoCbl, coenzyme B12) catalyzed by the ribonucleotide triphosphate reductase (RTPR) of Lactobacillus leichmannii has been studied by stopped-flow spectrophotometry. At a given temperature and constant concentration of AdoCbl, the observed rate constants, k obs, are essentially independent of the initial concentration of RTPR, but the spectral change, and hence the amount of cob(II)alamin formed at equilibrium, shows a hyperbolic dependence on [RTPR]0. This is interpreted as being a consequence of the known relatively weak binding of AdoCbl to the enzyme so that increasing enzyme concentration drives the binding equilibrium toward enzyme−coenzyme complex. Fitting the absorbance change data to a binding isotherm gives values of the binding equilibrium constant, K b, and the maximal absorbance change obtainable when the coenzyme is “saturated” with enzyme. From the latter value and the difference in molar absorptivity of AdoCbl and cob(II)alamin, the concentration of enzyme-bound cob(II)alamin, and consequently the equilibrium constant, K eq, for its formation from enzyme-bound AdoCbl, can be calculated. These values of K eq have been used to deconvolute the forward, k f, and reverse, k r, rate constants from the measured k obs values for cob(II)alamin formation at the active site. The enzyme is found to catalyze the Co−C bond homolysis of AdoCbl by a factor of 1.6 × 109 (ΔΔG ⧧ = 13 kcal mol-1) at 37 °C. Eyring plots of k f and van't Hoff plots of K eq both show distinct discontinuities at about 32 °C which may indicate the formation of an inactive, or less active, conformer at lower temperature or a change in mechanism. The activation parameters for the forward rate constant for cob(II)alamin in the upper temperature regime (ΔH f ⧧ = 20 ± 1 kcal mol-1, ΔS f ⧧ = 13 ± 4 cal mol-1 K-1) show that the entropy of activation is essentially the same as that for the nonenzymatic thermal homolysis of AdoCbl, but the enthalpy of activation is 13 ± 2 kcal mol-1 lower. The enzyme thus appears to catalyze the Co−C bond cleavage reaction entirely enthalpically. Several possibilities for the enthalpic catalysis of AdoCbl homolysis by RTPR are discussed.

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“…26, 27 The other important characteristic distinguishing the class I and class II enzymes is their specificity for AdoCbl. The class II enzymes, including ribonucleotide reductase, 28 diol dehydratase, 29 and glycerol dehydratase, 30 are remarkably promiscuous in tolerating structural alterations in the coenzyme. A large number of AdoCbl analogs with structural alterations in the adenine and ribose moieties, the corrin, and its side chains, and in the axial nucleotide are partially active coenzymes with these enzymes (although other AdoCbl analogs are inactive, and act as inhibitors).…”

Section: Introductionmentioning

confidence: 99%

The enzymatic activation of coenzyme B12

Brown

2006

Dalton Trans.

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The enzymatic "activation" of coenzyme B12 (5'-deoxyadenosylcobalamin, AdoCbl), in which homolysis of the carbon-cobalt bond of the coenzyme is catalyzed by some 10(9)- to 10(14)-fold, remains one of the outstanding problems in bioinorganic chemistry. Mechanisms which feature the enzymatic manipulation of the axial Co-N bond length have been investigated by theoretical and experimental methods. Classical mechanochemical triggering, in which steric compression of the long axial Co-N bond leads to increased upward folding of the corrin ring and stretching of the Co-C bond is found to be feasible by molecular modeling, but the strain induced in the Co-C bond seems to be too small to account for the observed catalytic power. The modeling study shows that the effect is a steric one which depends on the size of the axial nucleotide base, as substitution of imidazole (Im) for the normal 5,6-dimethylbenzimidazole (Bzm) axial base decreases the Co-C bond labilization considerably. An experimental test was thus devised using the coenzyme analog with Im in place of Bzm (Ado(Im)Cbl). Studies of the enzymatic activation of this analog by the B12-dependent ribonucleoside triphosphate reductase from Lactobacillus leichmannii coupled with studies of the non-enzymatic homolytic lability of the Co-C bond of Ado(Im)Cbl show that the enzyme is only slightly less efficient (3.8-fold, 0.8 kcal mol(-1)) at activating Ado(Im)Cbl than at activating AdoCbl itself. This suggests, in agreement with the modeling study, that mechanochemical triggering can make only a small contribution to the enzymatic activation of AdoCbl. Another possibility, electronic stabilization of the Co(II) homolysis product by compression of the axial Co-N bond, requires that enzymatic activation be sensitive to the basicity of the axial nucleotide. Preliminary studies of the enzymatic activation of a coenzyme analog with a 5-fluoroimidazole axial nucleotide suggest that the catalysis of Co-C bond homolysis may indeed be significantly slowed by the decrease in basicity.

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Study on the mechanism of action of adenosylcobalamin-dependent glycerol dehydratase from Aerobacter aerogenes

Cited by 27 publications

References 29 publications

Adocobinamide, the Base-off Analog of Coenzyme B₁₂ (Adocobalamin). 2.¹ Probing the “Base-on” Effect in Coenzyme B₁₂ via Cobalt−Carbon Bond Thermolysis Product and Kinetic Studies as a Function of Exogenous Pyridine Bases

Adocobinamide, the Base-off Analog of Coenzyme B₁₂ (Adocobalamin). 2.¹ Probing the “Base-on” Effect in Coenzyme B₁₂ via Cobalt−Carbon Bond Thermolysis Product and Kinetic Studies as a Function of Exogenous Pyridine Bases

Activation Parameters for the Carbon−Cobalt Bond Homolysis of Coenzyme B₁₂Induced by the B₁₂-Dependent Ribonucleotide Reductase fromLactobacillus leichmannii

The enzymatic activation of coenzyme B12

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Study on the mechanism of action of adenosylcobalamin-dependent glycerol dehydratase from Aerobacter aerogenes

Cited by 27 publications

References 29 publications

Adocobinamide, the Base-off Analog of Coenzyme B12 (Adocobalamin). 2.1 Probing the “Base-on” Effect in Coenzyme B12 via Cobalt−Carbon Bond Thermolysis Product and Kinetic Studies as a Function of Exogenous Pyridine Bases

Adocobinamide, the Base-off Analog of Coenzyme B12 (Adocobalamin). 2.1 Probing the “Base-on” Effect in Coenzyme B12 via Cobalt−Carbon Bond Thermolysis Product and Kinetic Studies as a Function of Exogenous Pyridine Bases

Activation Parameters for the Carbon−Cobalt Bond Homolysis of Coenzyme B12Induced by the B12-Dependent Ribonucleotide Reductase fromLactobacillus leichmannii

The enzymatic activation of coenzyme B12

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Product

Resources

About

Adocobinamide, the Base-off Analog of Coenzyme B₁₂ (Adocobalamin). 2.¹ Probing the “Base-on” Effect in Coenzyme B₁₂ via Cobalt−Carbon Bond Thermolysis Product and Kinetic Studies as a Function of Exogenous Pyridine Bases

Adocobinamide, the Base-off Analog of Coenzyme B₁₂ (Adocobalamin). 2.¹ Probing the “Base-on” Effect in Coenzyme B₁₂ via Cobalt−Carbon Bond Thermolysis Product and Kinetic Studies as a Function of Exogenous Pyridine Bases

Activation Parameters for the Carbon−Cobalt Bond Homolysis of Coenzyme B₁₂Induced by the B₁₂-Dependent Ribonucleotide Reductase fromLactobacillus leichmannii