| Autor: |
Bonvin, Julie, Aponte, Raphael A., Marcantonio, Maria, Singh, Sasha, Christendat, Dinesh, Turnbull, Joanne L. |
| Zdroj: |
Protein Science: A Publication of the Protein Society; 2006, Vol. 15 Issue 6, p1417-1432, 16p |
| Abstrakt: |
A monofunctional prephenate dehydrogenase (PD) from Aquifex aeolicus was expressed as a His-tagged protein in Escherichia coli and was purified by nickel affinity chromatography allowing the first biochemical and biophysical characterization of a thermostable PD. A. aeolicus PD is susceptible to proteolysis. In this report, the properties of the full-length PD are compared with one of these products, an N-terminally truncated protein variant (Δ19PD) also expressed recombinantly in E. coli. Both forms are dimeric and show maximum activity at 95°C or higher. Δ19PD is more sensitive to temperature effects yielding a half-life of 55 min at 95°C versus 2 h for PD, and values of kcat and Km for prephenate, which are twice those determined for PD at 80°C. Low concentrations of guanidine-HCl activate enzyme activity, but at higher concentrations activity is lost concomitant with a multi-state pathway of denaturation that proceeds through unfolding of the dimer, oligomerization, then unfolding of monomers. Measurements of steady-state fluorescence intensity and its quenching by acrylamide in the presence of Gdn-HCl suggest that, of the two tryptophan residues per monomer, one is buried in a hydrophobic pocket and does not become solvent exposed until the protein unfolds, while the less buried tryptophan is at the active site. Tyrosine is a feedback inhibitor of PD activity over a wide temperature range and enhances the cooperativity between subunits in the binding of prephenate. Properties of this thermostable PD are compared and contrasted with those of E. coli chorismate mutase-prephenate dehydrogenase and other mesophilic homologs. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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