EVOLUTION OF ENZYMATIC-ACTIVITIES IN THE ENOLASE SUPERFAMILY - PARTITIONING OF REACTIVE INTERMEDIATES BY (D)-GLUCARATE DEHYDRATASE FROM PSEUDOMONAS-PUTIDA

Glucarate dehydratase (GlucD) from Pseudomonas putida catalyzes the de hydration of both (D)-glucarate and (L)-idarate to 3-deoxy-(L)-threo-2 -hexulosarate as well as their epimerization. (D)-[6-C-13]Glucarate an d (L)-[6-C-13]idarate have been synthesized for use in continuous assa y of the reactions catalyzed by GlucD by both C-13 and H-1 NMR spectro scopies, thereby allowing the simultaneous measure of both the dehydra tion and epimerization reactions. Substrate and solvent isotope effect s for the dehydration reactions have been quantitated. The mechanism o f the GlucD-catalyzed reaction is discussed in the context of that pre viously established for the homologous mandelate racemase from P. puti da, also a member of the enolase superfamily whose members catalyze re actions initiated by abstraction of a proton a to a carboxylate group.