Probing the cob(II)alamin Cond UctorHhypothesis with Glutamate Mutase from Clostridium Cochlearium

Abstract

It had been proposed that during reversible coenzyme B₁₂ dependent rearrangements, cob(II)alamin is not merely present as a spectator but also acts as a conductor by stabilizing the methylene radical intermediates. Density functional theory (DFT) calculations suggested a hydrogen bond between C₁₉-H of the corrin ring and the 3'-OH moiety (O3RL) of the 5'- deoxyadenosyl radical resulting in a decrease of the activation energy by about 30 kJ mol^-1. We tested this hypothesis with glutamate mutase and artificial coenzyme B₁₂ derivatives. The assembly of coenzyme B₁₂ (adenosylcobalamin) with recombinant components GlmS and GlmE of glutamate mutase from Clostridium cochlearium reconstitutes an active holoenzyme that catalyses the reversible rearrangement between (S)-glutamate and (2S,3S)-3-methylaspartate. Glutamate mutase activity was also demonstrated upon incubation of GlmS and E with 3',5'- dideoxyadenosylcobalamin, but not with 2',5'-dideoxyadenosylcobalamin and peptidoadenylcobalamin. In the latter cobalamin, the ribose unit of the upper ligand was replaced with a peptide mimic that contains the same number of atoms between Co(III) and the adenosine base. Measurements of the kinetic constants of glutamate mutase with coenzyme B₁₂ and 3',5'- dideoxyadenosylcobalamin suggested similar binding properties of the cofactors to the apoenzyme. However, the catalytic efficiency (k_cat/K_m) of glutamate mutase was 15 times reduced with 3',5'-dideoxyadenosylcobalamin compared to catalysis with coenzyme B₁₂ as cofactor. This translates into a stabilization of only 7 kJ mol^-1 in the substrate activation step. We attribute this effect to weak interactions of O3' of the riboise moiety with either C₁₉-H of the corrin ring or with the glutamate residue 330 of component E (Glu330). The catalytic inactivity of 2',5'- dideoxyadenosylcobalamin and peptidoadenylcobalamin reveals critical interactions of the 2'-OH moiety (O2') during the catalytic cycle. Evidence for H-bonding between O2' and Glu330 is obtained from the crystal structure analysis of glutamate mutase’s active site.