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A conformational sampling model for radical catalysis in pyridoxal phosphate- and cobalamin-dependent enzymes.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2014 Dec 05; Vol. 289 (49), pp. 34161-74. Date of Electronic Publication: 2014 Sep 11. - Publication Year :
- 2014
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Abstract
- Cobalamin-dependent enzymes enhance the rate of C-Co bond cleavage by up to ∼10(12)-fold to generate cob(II)alamin and a transient adenosyl radical. In the case of the pyridoxal 5'-phosphate (PLP) and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5 aminomutase (OAM), it has been proposed that a large scale domain reorientation of the cobalamin-binding domain is linked to radical catalysis. Here, OAM variants were designed to perturb the interface between the cobalamin-binding domain and the PLP-binding TIM barrel domain. Steady-state and single turnover kinetic studies of these variants, combined with pulsed electron-electron double resonance measurements of spin-labeled OAM were used to provide direct evidence for a dynamic interface between the cobalamin and PLP-binding domains. Our data suggest that following ligand binding-induced cleavage of the Lys(629)-PLP covalent bond, dynamic motion of the cobalamin-binding domain leads to conformational sampling of the available space. This supports radical catalysis through transient formation of a catalytically competent active state. Crucially, it appears that the formation of the state containing both a substrate/product radical and Co(II) does not restrict cobalamin domain motion. A similar conformational sampling mechanism has been proposed to support rapid electron transfer in a number of dynamic redox systems.<br /> (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Subjects :
- Amino Acid Sequence
Bacterial Proteins genetics
Bacterial Proteins metabolism
Biocatalysis
Clostridium sticklandii enzymology
Escherichia coli genetics
Escherichia coli metabolism
Free Radicals chemistry
Free Radicals metabolism
Hydrophobic and Hydrophilic Interactions
Intramolecular Transferases genetics
Intramolecular Transferases metabolism
Kinetics
Lysine chemistry
Lysine metabolism
Molecular Conformation
Molecular Dynamics Simulation
Molecular Sequence Data
Mutagenesis, Site-Directed
Ornithine chemistry
Ornithine metabolism
Pyridoxal Phosphate metabolism
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Static Electricity
Vitamin B 12 metabolism
Bacterial Proteins chemistry
Clostridium sticklandii chemistry
Intramolecular Transferases chemistry
Pyridoxal Phosphate chemistry
Vitamin B 12 chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 289
- Issue :
- 49
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 25213862
- Full Text :
- https://doi.org/10.1074/jbc.M114.590471