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Mechanistic studies on chorismate mutase-prephenate dehydrogenase from E. coli

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Mechanistic studies on chorismate mutase-prephenate dehydrogenase from E. coli

Christendat, Dinesh Singh (1998) Mechanistic studies on chorismate mutase-prephenate dehydrogenase from E. coli. PhD thesis, Concordia University.

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Abstract

Chorismate mutase-prephenate dehydrogenase is a bifunctional enzyme that catalyzes two sequential reactions in the biosynthesis of tyrosine in E. coli and other enteric bacteria. Chorismate mutase catalyzes the pericyclic rearrangement of chorismate to prephenate which is subsequently converted to (4-hydroxyphenyl)pyruvate through an oxidative decarboxylation reaction catalyzed by prephenate dehydrogenase. Through chemical modification and site-directed mutagenesis we have identified residues that are critical in both the mutase and dehydrogenase mechanisms and have provided evidence that these two reactions occur at separate active sites on the enzyme. We have identified Lys37 as a residue that is important in the mutase reaction by differential peptide mapping. This result was confirmed by site-directed mutagenesis. The crystal structures of other chorismate mutases indicate that Lys37 may provide important hydrogen bonds in the transition state of the mutase reaction. We chemically modified mutase-dehydrogenase to show that a histidine is important for dehydrogenase activity. The pH rate profile for the dehydrogenase reaction indicates that a protonated residue is important for catalysis. By comparing the pH profile for wild-type and mutant mutase-dehydrogenase we have identified His197 as this catalytic group. Sequence alignments with other prephenate dehydrogenase enzymes show that this histidine is highly conserved. We propose that His197 may be the catalytic base involved in the hydride transfer from prephenate to NAD + . We have identified three positively charged residues, Lys178, Arg286 and Arg294, that are conserved amongst prephenate dehydrogenases from different organisms. We conducted site-directed mutagenesis on these residues and compared the wild-type enzyme and selected mutants with respect to their stability, pH rate profiles and ability to bind prephenate and a series of inhibitory substrate analogues. Our results indicate that Arg294 plays an important role in prephenate binding by interacting electrostatically with the ring carboxylate of the substrate. Our studies also show that a group with a p K of 8.8 interacts with the pyruvyl side chain carboxylate of prephenate. Further characterization of mutant proteins is being conducted to determine if this binding group is either Lys178 or Arg286

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (PhD)
Authors:Christendat, Dinesh Singh
Pagination:xix, 209 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:Theses (Ph.D.)
Program:Chemistry and Biochemistry
Date:1998
Thesis Supervisor(s):Turnbull, Joanne
ID Code:520
Deposited By:Concordia University Libraries
Deposited On:27 Aug 2009 13:12
Last Modified:08 Dec 2010 10:15
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