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 Hisα143. Mutant enzymes in which Hisα143 was mutated to another amino acid residue were expressed in Escherichia coli, purified, and examined for enzymatic activity. The Hα143Q mutant was 34% as active as the wild-type enzyme. Hα143A and Hα143L 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 α143 is important not only for catalysis but also for protecting radical intermediates. Hα143E and Hα143K that did not exist as (αβγ)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 Hα143Q and partially rate-determining for Hα143A. The preference for substrate enantiomers was reversed by the Hα143Q mutation in both substrate binding and catalysis. Upon the inactivation of the Hα143A 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 Hα143Q underwent irreversible inactivation by O2 in the absence of substrate at a much lower rate than the wild type.
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