Dataset Information

Dioldehydrase: an essential role for potassium ion in the homolytic cleavage of the cobalt-carbon bond in adenosylcobalamin.

ABSTRACT: The complex of dioldehydrase with adenosylcobalamin (coenzyme B12) and potassium ion reacts with molecular oxygen in the absence of a substrate to oxidize coenzyme and inactivate the complex. In this article, high performance liquid chromatography and mass spectral analysis are used to identify the nucleoside products resulting from oxygen inactivation. The product profile indicates that oxygen inactivation proceeds by direct reaction of molecular oxygen with the 5'-deoxyadenosyl radical and cob(II)alamin. Formation of 5'-peroxyadenosine as the initial nucleoside product chemically correlates this reaction with aerobic, aqueous photoinduced homolytic cleavage of adenosylcobalamin (Schwartz, P. A., and Frey, P. A., (2007) Biochemistry, in press), indicating that both reactions proceed through similar chemical intermediates. The oxygen inactivation of the enzyme-coenzyme complex shows an absolute requirement for the same monocations required in catalysis by dioldehydrase. Measurements of the dissociation constants for adenosylcobalamin from potassium-free (Kd = 16 +/- 2 microM) or potassium-bound dioldehydrase (Kd = 0.8 +/- 0.2 microM) reveal that the effect of the monocation in stimulating oxygen sensitivity cannot be explained by an effect on the binding of coenzyme to the enzyme. Cross-linking experiments suggest that the full quaternary structure is assembled in the absence of potassium ion under the experimental conditions. The results indicate that dioldehydrase likely harvests the binding energy of the activating monocation to stimulate the homolytic cleavage of the Co-C5' bond in adenosylcobalamin.

SUBMITTER: Schwartz PA

PROVIDER: S-EPMC2516201 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Dioldehydrase: an essential role for potassium ion in the homolytic cleavage of the cobalt-carbon bond in adenosylcobalamin.

Schwartz Phillip A PA Frey Perry A PA

Biochemistry 20070522 24

The complex of dioldehydrase with adenosylcobalamin (coenzyme B12) and potassium ion reacts with molecular oxygen in the absence of a substrate to oxidize coenzyme and inactivate the complex. In this article, high performance liquid chromatography and mass spectral analysis are used to identify the nucleoside products resulting from oxygen inactivation. The product profile indicates that oxygen inactivation proceeds by direct reaction of molecular oxygen with the 5'-deoxyadenosyl radical and cob ...[more]

PMID: 17516630

Similar Datasets

Project description:Protein contributions to the substrate-triggered cleavage of the cobalt-carbon (Co-C) bond and formation of the cob(II)alamin-5'-deoxyadenosyl radical pair in the adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been studied by using pulsed-laser photolysis of AdoCbl in the EAL-AdoCbl-substrate ternary complex, and time-resolved probing of the photoproduct dynamics by using ultraviolet-visible absorption spectroscopy on the 10(-7)-10(-1) s time scale. Experiments were performed in a fluid dimethylsulfoxide/water cryosolvent system at 240 K, under conditions of kinetic competence for thermal cleavage of the Co-C bond in the ternary complex. The static ultraviolet-visible absorption spectra of holo-EAL and ternary complex are comparable, indicating that the binding of substrate does not labilize the cofactor cobalt-carbon (Co-C) bond by significantly distorting the equilibrium AdoCbl structure. Photolysis of AdoCbl in EAL at 240 K leads to cob(II)alamin-5'-deoxyadenosyl radical pair quantum yields of <0.01 at 10(-6) s in both holo-EAL and ternary complex. Three photoproduct states are populated following a saturating laser pulse, and labeled, P(f), P(s), and P(c). The relative amplitudes and first-order recombination rate constants of P(f) (0.4-0.6; 40-50 s(-1)), P(s) (0.3-0.4; 4 s(-1)), and P(c) (0.1-0.2; 0) are comparable in holo-EAL and in the ternary complex. Time-resolved, full-spectrum electron paramagnetic resonance (EPR) spectroscopy shows that visible irradiation alters neither the kinetics of thermal cob(II)alamin-substrate radical pair formation, nor the equilibrium between ternary complex and cob(II)alamin-substrate radical pair, at 246 K. The results indicate that substrate binding to holo-EAL does not "switch" the protein to a new structural state, which promptly stabilizes the cob(II)alamin-5'-deoxyadenosyl radical pair photoproduct, either through an increased barrier to recombination, a decreased barrier to further radical pair separation, or lowering of the radical pair state free energy, or a combination of these effects. Therefore, we conclude that such a change in protein structure, which is independent of changes in the AdoCbl structure, and specifically the Co-C bond length, is not a basis of Co-C bond cleavage catalysis. The results suggest that, following the substrate trigger, the protein interacts with the cofactor to contiguously guide the cleavage of the Co-C bond, at every step along the cleavage coordinate, starting from the equilibrium configuration of the ternary complex. The cleavage is thus represented by a diagonal trajectory across a free energy surface, that is defined by chemical (Co-C separation) and protein configuration coordinates.

Dataset Information

Dioldehydrase: an essential role for potassium ion in the homolytic cleavage of the cobalt-carbon bond in adenosylcobalamin.

Publications

Dioldehydrase: an essential role for potassium ion in the homolytic cleavage of the cobalt-carbon bond in adenosylcobalamin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets