The His143, Glu170, and Asp335 residues at the substrate-binding site of diol dehydratase, a calcium-metalloenzyme, are shown by a computational mutation study to play important roles in OH group migration (the second step in the enzymatic reaction). The reaction is accelerated by the synergetic interplay of the heterolysis of the C2-O2 bond of 1,2-diol radical and the partial deprotonation of the spectator OH group by Glu170. The His143 residue works as a donor to the migrating OH group through a hydrogen bond, which contributes to the C2-O2 bond heterolysis and resultant resonance stabilization. The Glu170 residue activates the spectator OH group to energetically stabilize the transition state in the OH group migration. The resonance stabilization of the transition state in the OH group migration is observed in the wild-type enzyme while not in the His143Ala mutant. Since the cleavage of the C2-O2 bond of 1,2-diol radical proceeds in a more homolytic manner in the His143Ala mutant, Glu170 cannot effectively deprotonate the spectator OH group in the transition state. As a result, the activation energy of the OH group migration in the His143Ala mutant is increased compared to that in the wild-type enzyme. The spectator OH group is not fully activated in the Glu170Gln and Glu170Ala mutants during the OH group migration, and thus the activation energies in the Glu170Gln and Glu170Ala mutants are higher than that in the wild-type enzyme. In contrast, the OH group migration is accelerated in the Asp335Ala mutant, due to the absence of the electric repulsion between Asp335 and the migrating OH group. The computed relative activity of the His143Ala, Glu170Gln, and Glu170Ala mutants successfully reproduces the experimentally determined catalytic activity, indicating that a computational mutation study offers a useful methodology in enzyme research.
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