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 dehydration of both (D)-glucarate and (L)-idarate to 3-deoxy-(L)-threo-2-hexulosarate as well as their epimerization. (D)-[6-13C]Glucarate and (L)-[6-13C]idarate have been synthesized for use in continuous assay of the reactions catalyzed by GlucD by both 13C and 1H NMR spectroscopies, thereby allowing the simultaneous measure of both the dehydration and epimerization reactions. Substrate and solvent isotope effects for the dehydration reactions have been quantitated. The mechanism of the GlucD-catalyzed reaction is discussed in the context of that previously established for the homologous mandelate racemase from P. putida, also a member of the enolase superfamily whose members catalyze reactions initiated by abstraction of a proton alpha to a carboxylate group.
Palmer
D R
DR
Department of Biochemistry, University of Illinois at Urbana-Champaign 61801, USA.
Hubbard
B K
BK
Gerlt
J A
JA
D002247:Carbon Isotopes; D002384:Catalysis; D003903:Deuterium; D004735:Energy Transfer; D004789:Enzyme Activation; D019143:Evolution, Molecular; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D016958:Pseudomonas putida / Q000201:enzymology*; D012997:Solvents; D013237:Stereoisomerism; D013379:Substrate Specificity
D002247:Carbon Isotopes; D011522:Protons; D012997:Solvents; D003903:Deuterium; D006836:Hydro-Lyases; D010751:Phosphopyruvate Hydratase; C020572:glucarate dehydratase