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Probing the importance of active site residues of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus

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Probing the importance of active site residues of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus

Hou, Wenjuan (2009) Probing the importance of active site residues of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. Masters thesis, Concordia University.

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Abstract

Prephenate dehydrogenase is an enzyme within the TyrA protein family dedicated to tyrosine biosynthesis. It catalyzes the oxidative decarboxylation of prephenate to hydroxyphenylpyruvate (HPP) and CO 2 in the presence of NAD + ; HPP is then transaminated to L-tyrosine, an end product inhibitor of the pathway. Guided by the recently published crystal structures of a monofunctional prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus , amino acid residues Trp190, His217, Lys246' and Arg250 were targeted for mutagenesis in order to provide insights into how these side chains might participate in the chemistry of the reaction and contribute to the integrity of the active site. A revised purification protocol afforded excellent yields of variants. We identified Trp190, a completely buried side chain in the NAD + binding pocket, as the source of an unusual fluorescence emission peak at 317 nm. Analysis of variants of Lys246' and Arg250 allowed us to conclude that the two residues participated only in the binding of prephenate and not of NAD + or in catalysis. They are important residues but not essential for binding. In contrast, His217 is critical for prephenate binding and catalysis and for the inhibition of PD activity by tyrosine. Chemical and thermal denaturation studies also suggest that this residue helps to maintain the structural integrity of the active site. Our results are interpreted in light of the crystal structure of the enzyme bound with substrate analogues and tyrosine.

Divisions:Concordia University > Faculty of Arts and Science > Biology
Item Type:Thesis (Masters)
Authors:Hou, Wenjuan
Pagination:xi, 96 leaves : ill. (some col.) ; 29 cm.
Institution:Concordia University
Degree Name:M. Sc.
Program:Biology
Date:2009
Thesis Supervisor(s):Turnbull, Joanne
ID Code:976372
Deposited By: Concordia University Library
Deposited On:22 Jan 2013 16:24
Last Modified:18 Jan 2018 17:42
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