Roles of adenine anchoring and ion pairing at the coenzyme B ₁₂-binding site in diol dehydratase catalysis

The X-ray structure of the diol dehydratase-adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Serα224, and of 6-NH₂, N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the ε-NH₂ group of Lysβ135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Serα224 and Lysβ135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Sα224A, Sα224N and Kβ135E mutants were 19-2% as active as the wild-type enzyme, whereas the Kβ135A, Kβ135Q and Kβ135R mutants retained 58-76% of the wild-type activity. The presence of a positive charge at the β135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme-cobalamin interaction. The Sα224A and Sα224N mutants showed a k _cat/k_inact value that was less than 2% that of the wild-type, whereas for Lysβ135 mutants this value was in the range 25-75%, except for the Kβ135E mutant (7%). Unlike the wild-type holoenzyme, the Sα224N and Sα224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Serα224 is important for cobalt-carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Sα224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5′-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one.