TY - JOUR
T1 - A conformational sampling model for radical catalysis in pyridoxal phosphate- and cobalamin-dependent enzymes
AU - Menon, Binuraj R. K.
AU - Fisher, Karl
AU - Rigby, Stephen E. J.
AU - Scrutton, Nigel S.
AU - Leys, David
N1 - Publisher Copyright:
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2014/12/5
Y1 - 2014/12/5
N2 - 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.
AB - 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.
KW - Adenosylcobalamin (AdoCbl)
KW - Electron Paramagnetic Resonance (EPR)
KW - protein dynamic
KW - pyridoxal phosphate
KW - radical
KW - conformational sampling
KW - domain dynamics
KW - ornithine aminomutase
UR - http://www.scopus.com/inward/record.url?scp=84917707289&partnerID=8YFLogxK
U2 - 10.1074/jbc.M114.590471
DO - 10.1074/jbc.M114.590471
M3 - Article
C2 - 25213862
AN - SCOPUS:84917707289
SN - 0021-9258
VL - 289
SP - 34161
EP - 34174
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 49
ER -