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Mechanism-based Inhibition Reveals Transitions between Two Conformational States in the Action of Lysine 5,6-Aminomutase: A Combination of Electron Paramagnetic Resonance Spectroscopy, Electron Nuclear Double Resonance Spectroscopy, and Density Functional Theory Study
An “open”-state crystal structure of lysine
5,6-aminomutase
suggests that transition to a hypothetical “closed”-state
is required to bring the cofactors adenosylcobalamin (AdoCbl) and
pyridoxal-5′-phosphate (PLP) and the substrate into proximity
for the radical-mediated 1,2-amino group migration. This process is
achieved by transaldimination
of the PLP–Lys144β internal aldimine with the PLP–substrate
external aldimine. A closed-state crystal structure is not available.
UV–vis and electron paramagnetic resonance studies show that
homologues of substrate d-lysine, 2,5-DAPn, 2,4-DAB, and
2,3-DAPr bind to PLP as an external aldimine and elicit the AdoCbl
Co–C bond homolysis and the accumulations of cobÂ(II)Âalamin
and analogue-based radicals, demonstrating the existence of a closed
state. <sup>2</sup>H- and <sup>31</sup>P-electron nuclear double resonance
studies, supported by computations, show that the position for hydrogen
atom abstraction from 2,5-DAPn and 2,4-DAB by the 5′-deoxyadenosyl
radical occurs at the carbon adjacent to the imine, resulting in overstabilized
radicals by spin delocalization through the imine into the pyridine
ring of PLP. These radicals block the active site, inhibit the enzyme,
and poise the enzyme into two distinct conformations: for even-numbered
analogues, the cobÂ(II)Âalamin remains proximal to and spin-coupled
with the analogue-based radical in the closed state while odd-numbered
analogues could trigger the transition to the open state of the enzyme.
We provide here direct spectroscopic evidence that strongly support
the existence of a closed state and its analogue-dependent transition
to the open state, which is one step that was proposed to complete
the catalytic turnover of the substrate lysine