Galactonate Dehydratase: Exploring the Frontiers of the Enolase Superfamily
The crystal structure of GalD indicates that His 185 may serve as a general acid catalyst to facilitate the departure of the 3-OH leaving group and/or stereospecifically protonate the product in the subsequent tautomerization reaction. The function of His 185 was investigated by comparing the kinetics of wild type, H285N, E310Q, H185Q and H185N GalD mutants, using both D-galactonate and 3-fluoro-3-deoxy-galactonate (F-Gal) as substrates. The kcat values for all the mutants decreased with D-galactonate, but only the kcat values for H185Q and H185N were relatively unaffected with F-Gal. Solvent deuterium incorporation studies with H185Q and H185N indicated that deuterium is stereospecifically protonated at the pro-S position. Thus, His 185 is involved in the beta-elimination of 3-OH but does not protonate the product in the subsequent tautomerization reaction.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign