A conformational sampling model for radical catalysis in pyridoxal phosphate- and cobalamin-dependent enzymes.
A conformational sampling model for radical catalysis in pyridoxal phosphate- and cobalamin-dependent enzymes.
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DOI：
10.1074/jbc.m114.590471
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发表时间：
2014-12-05
期刊：
影响因子：
--
通讯作者：
Leys D
中科院分区：
文献类型：
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作者：
Menon BR;Fisher K;Rigby SE;Scrutton NS;Leys D
Background: Catalysis in ornithine 4,5-aminomutase (OAM) may involve large scale domain dynamics. Results: A conformational equilibrium is established following release of Lys629 from the PLP cofactor. Conclusion: Substrate binding triggers conformational sampling of the cobalamin-binding domain to support catalysis. Significance: A model for radical catalysis involving domain conformational sampling is established for pyridoxal phosphate/cobalamin-dependent enzymes. Cobalamin-dependent enzymes enhance the rate of C–Co bond cleavage by up to ∼1012-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 Lys629-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.
