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A conformational sampling model for radical catalysis in pyridoxal phosphate- and cobalamin-dependent enzymes.

The Journal of biological chemistry (2014-09-13)
Binuraj R K Menon, Karl Fisher, Stephen E J Rigby, Nigel S Scrutton, David Leys
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Vitamin B12, ≥98%
Sigma-Aldrich
L-Lysine, ≥98% (TLC)
Supelco
Cyanocobalamin, pharmaceutical secondary standard, certified reference material
Sigma-Aldrich
Vitamin B12, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥98%
Sigma-Aldrich
L-Lysine monohydrochloride, reagent grade, ≥98% (HPLC)
Supelco
Cyanocobalamin (B12), analytical standard
Sigma-Aldrich
L-Ornithine monohydrochloride, ≥99%
Sigma-Aldrich
L-Lysine monohydrochloride, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
L-Lysine, crystallized, ≥98.0% (NT)
Supelco
L-Lysine monohydrochloride, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Cyanocobalamin, meets USP testing specifications
Supelco
L-Lysine, analytical standard
Cyanocobalamin, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
L-Lysine monohydrochloride, BioUltra, ≥99.5% (AT)
Sigma-Aldrich
L-Ornithine monohydrochloride, BioReagent, suitable for cell culture, ≥99%
Lysine hydrochloride, European Pharmacopoeia (EP) Reference Standard
Supelco
L-Lysine monohydrochloride, certified reference material, TraceCERT®
Sigma-Aldrich
Cyanocobalamin, tested according to Ph. Eur.
Sigma-Aldrich
L-Ornithine hydrochloride, 99%
Sigma-Aldrich
L-Lysine acetate salt, ≥98% (HPLC)
Sigma-Aldrich
D-Ornithine monohydrochloride, ~98%
Supelco
L-Lysine Acetate, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
L-Ornithine monohydrochloride, BioXtra, ≥99%
Sigma-Aldrich
L-Ornithine monohydrochloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
L-Ornithine dihydrochloride, ≥99.0% (AT)
Sigma-Aldrich
DL-Ornithine monohydrochloride, ≥99.0% (AT)
Lysine acetate, European Pharmacopoeia (EP) Reference Standard