A Conformational Sampling Model for Radical Catalysis in Pyridoxal Phosphate- and Cobalamin-dependent Enzymes [Protein Structure and Folding]

September 11th, 2014 by Menon, B. R. K., Fisher, K., Rigby, S. E. J., Scrutton, N. S., Leys, D.

Cobalamin-dependent enzymes enhance the rate of C-Co bond cleavage by up to ca 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 (LAM) 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 (PELDOR) measurements of spin-labelled 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.
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