Production and purification of imidazole glycerol phosphate dehydratase from Escherichia coli and Cryptococcus neoformans: Comparative kinetic and metal binding studies
Abstract
Imidazole glycerol phosphate dehydratase (IGPD) is a metal dependent enzyme that catalyzes the dehydration of IGP to imidazole acetol phosphate (IAP) in the histidine biosynthetic pathway of microorganisms and plants. IGPD from Escherichia coli is bifunctional and also exhibits histidinol phosphate phosphatase activity, while the enzyme from Cryptococcus neoformans has only IGPD activity. The IGPD reaction poses many interesting mechanistic questions, many of which are related to the fact that IGP is unusual in comparison to most substrates for enzyme catalyzed dehydrations. Stereochemical analyses on E. coli IGPD revealed that no intramolecular hydride, proton or hydrogen atom transfer occurs during the dehydration, and IGPD's exact mechanism of action remains unknown. Many enzymes that catalyze dehydrations utilize metal ions, either as Lewis acids or to increase the nucleophilicity of reactive water molecules in the active site. In order to learn more about the IGPD reaction, a comparative investigation of the basic biochemical and metal binding characteristics of IGPD from E. coli and C. neoformans was initiated. Protein production and purification systems for both IGPDs were developed, and a continuous spectrophotometric assay was designed and used to determine the basic kinetic characteristics of each protein. Enzymatic substrate preparation protocols were created. EPR studies and enzymatic assays demonstrated that both proteins have optimal activity when charged with one manganese (II) ion per subunit ($\rm K\sb{d}={\sim1}\ \mu M).$ Other metal ions can activate the proteins; however, metal ion preferences of E. coli and C. neoformans IGPDs vary slightly. Crystallographic studies on C. neoformans IGPD were initiated and yielded cubic crystals which diffract to 5 A resolution. The product of the IGPD reaction, IAP, exhibits unusual solution chemistry, atypical of ketones, and this behavior was examined using NMR, EPR and UV-visible spectroscopy.
Degree
Ph.D.
Advisors
Schwab, Purdue University.
Subject Area
Biochemistry
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