Glutamate 338 is an electrostatic facilitator of C-Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase.

How cobalamin-dependent enzymes promote C-Co homolysis to initiate radical catalysis has been debated extensively. For the pyridoxal 5'-phosphate and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5-aminomutase (OAM), large-scale re-orientation of the cobalamin-binding domain linked to C-Co bond breakage has been proposed. In these models, substrate binding triggers dynamic sampling of the B12 -binding Rossmann domain to achieve a catalytically competent 'closed' conformational state. In 'closed' conformations of OAM, Glu338 is thought to facilitate C-Co bond breakage by close association with the cobalamin adenosyl group. We investigated this using stopped-flow continuous-wave photolysis, viscosity dependence kinetic measurements, and electron paramagnetic resonance spectroscopy of a series of Glu338 variants. We found that substrate-induced C-Co bond homolysis is compromised in Glu388 variant forms of OAM, although photolysis of the C-Co bond is not affected by the identity of residue 338. Electrostatic interactions of Glu338 with the 5'-deoxyadenosyl group of B12 potentiate C-Co bond homolysis in 'closed' conformations only; these conformations are unlocked by substrate binding. Our studies extend earlier models that identified a requirement for large-scale motion of the cobalamin domain. Our findings indicate that large-scale motion is required to pre-organize the active site by enabling transient formation of 'closed' conformations of OAM. In 'closed' conformations, Glu338 interacts with the 5'-deoxyadenosyl group of cobalamin. This interaction is required to potentiate C-Co homolysis, and is a crucial component of the approximately 10(12) rate enhancement achieved by cobalamin-dependent enzymes for C-Co bond homolysis.