Histidine-α143 assists 1,2-hydroxyl group migration and protects radical intermediates in coenzyme B12-dependent diol dehydratase

Diol dehydratase of Klebsiella oxytoca contains an essential histidine residue. Its X-ray structure revealed that the migrating hydroxyl group on C2 of substrate is hydrogen-bonded to His alpha 143. Mutant enzymes in which His alpha 143 was mutated to another amino acid residue were expressed in Escherichia coli, purified, and examined for enzymatic activity. The H alpha 143Q mutant was 34% as active as the wildtype enzyme. H alpha 143A and H alpha 143L showed only a trace of activity. Kinetic analyses indicated that the hydrogen bonding interaction between the hydroxyl group on C2 of substrate and the side chain of residue alpha 143 is important not only for catalysis but also for protecting radical intermediates. H alpha 143E and H alpha 143K that did not exist as (alpha beta gamma)(2) complexes were inactive. The deuterium kinetic isotope effect on the overall reaction suggested that a hydrogen abstraction step is fully rate-determining for the wild type and H alpha 143Q and partially rate-determining for H alpha 143A. The preference for substrate enantiomers was reversed by the H alpha 143Q mutation in both substrate binding and catalysis. Upon the inactivation of the H alpha 143A holoenzyme by 1,2-propanediol, cob(II)alamin without an organic radical coupling partner accumulated, 5'-deoxyadenosine was quantitatively formed from the coenzyme adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus concluded to be a mechanism-based inactivation. The holoenzyme of H alpha 143Q underwent irreversible inactivation by O-2 in the absence of substrate at a much lower rate than the wild type.