Hotz, Natascha (2010) Active Site Studies of a Thermophilic Dehydrogenase. Masters thesis, Concordia University.
- Accepted Version
Prephenate dehydrogenase (PD) is one enzyme within the family of TyrA proteins dedicated to the biosynthesis of L-tyrosine (L-Tyr). It catalyzes the NAD+-dependent oxidative decarboxylation of prephenate to hydroxyphenylpyruvate (HPP), which then undergoes transamination to L-Tyr, a feedback inhibitor of the enzyme. Guided by the recent crystal structures of a monofunctional PD from the hyperthermophilic bacterium Aquifex aeolicus in complex with active site ligands (63), residues were targeted for mutagenesis and the variant proteins were characterized by kinetic, biophysical and computational methods as an attempt to provide insight into how these residues participate in the catalytic mechanism and mode of regulation of A. aeolicus PD. We identified H205, a highly conserved residue, as an important catalytic group which likely maintains H147 in a catalytically competent conformation via a hydrogen bonding network, whereas electrostatic interactions afforded by D206 appear critical for the overall stability of the enzyme. We demonstrated that S254 is critical for feedback inhibition by L-Tyr, likely mediated by a hydrogen bonding network involving S254, T152, H217 and water. Moreover, R250 and K246 act in an additive fashion to facilitate the binding of prephenate to the enzyme. The combination of substrates and L-Tyr with the enzyme was further probed using fluorescence emission quenching, equilibrium dialysis and isothermal titration microcalorimetry. The latter technique revealed that L-Tyr binds with very high affinity to the native enzyme and that the combination is enthalpy driven. Perturbations in the binding of L-Tyr are further highlighted for selected variant proteins.
|Divisions:||Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry|
|Item Type:||Thesis (Masters)|
|Degree Name:||M. Sc.|
|Deposited By:||NATASCHA HOTZ|
|Deposited On:||13 Jun 2011 13:13|
|Last Modified:||01 Jan 2013 06:38|
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