Folding and stability of shikimate kinase

Cerasoli, Eleonora (2002) Folding and stability of shikimate kinase. PhD thesis, University of Glasgow.

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Shikimate kinase (SK; EC, an enzyme that catalyses the specific phosphorylation of the 3-hydroxyl group of shikimate using ATP as the phosphoryl donor, was chosen as a representative example of a subclass of a/p proteins with which to examine the unfolding, stability and mechanism of protein folding. The equilibrium unfolding of the enzyme was monitored for SK in the absence and in the presence of shikimate and using both guanidinium chloride (GdmCl) and urea as denaturing agents. The changes in secondary and tertiary structure, monitored by far UV, circular dichroism (CD) and fluorescence respectively, could be fitted for both denaturants to a 2-state model giving an estimate of 17 kJ/mol as the stability of the folded state. The marked loss of activity and decrease in shikimate binding observed at low concentrations of GdmCI were found to be largely due to ionic strength effects associated with perturbation of the shikimate binding site. The effects of different salts (NaCl, KCl, CaCl2, MgCl2, NaF, NaBr, NaNO3 and Na2S04) on the structure, activity, binding, and stability of SK have been investigated. The inclusion of salts leads to a marked stabilisation against unfolding of the enzyme by urea. Although the salts listed above have only small effects on far-UV CD and fluorescence spectra, NaCI and Na2S04 were found to lead to a tightening of the structure of the enzyme as shown by the effect on the near-UV CD spectrum. All the salts studied have an effect on catalytic activity, which in general appears to be correlated with changes in ionic strength. The refolding of SK after chemical denaturation using both urea and GdmCI has been studied in detail. This was performed using measurements of CD, fluorescence, activity, 8-anilino-1-naphthalene sulphonate (ANS) fluorescence, and employing both manual mixing and rapid reaction techniques. From these studies an outline pathway for the folding process has been formulated in which at least three intermediates are involved. The pathway described could act as a model for other members of the NMP kinase family of enzymes and the relevance of the results to the folding of other alpha/beta domain proteins is discussed. The unfolding of the enzyme by 4M urea could be prevented by inclusion of 1M NaCl or 0.33M Na2S04, and the addition of these concentrations of salts to enzyme unfolded by 4M urea led to a slow but nearly complete regain of overall native secondary and tertiary structure, as judged by the regain of native-like CD and fluorescence. However, the refolded enzyme differs from the native form in terms of the degree of fluorescence quenching by iodide ions which suggests that the single Trp has only partially regained the positively charged environment provided by neighbouring Arg side chains and in terms of the relatively weak binding of shikimate that can be observed by fluorescence quenching. While no binding of nucleotide could be detected directly there is some evidence for synergism in substrate binding which suggests that nucleotide may bind to some extent. However, the refolded enzyme does not possess detectable catalytic activity. During the refolding process brought about by addition of salt in the presence of 4M urea no change in the fluorescence of the probe ANS was observed, indicating that either an intermediate formed by hydrophobic collapse is unlikely to be significantly populated or that the concentration of the chaotropic agent used for denaturation displaces the dye by shear competition. The results point to both specific and general effects of salts on SK and are discussed in the light of the structural information available on the enzyme. Additional experiments to investigate the effect of the residual ionic strength on the process of refolding after a denaturant jump were undertaken. In general the presence of NaCI and Na2S04 lead to an increase in the refolding rate, while NaF seems to impair the efficiency of refolding. Preliminary experiments to investigate the protection by substrates against inactivation of SK by the lysine-specific reagent pyridoxal-5'-phosphate (PLP) were carried out. The inclusion of shikimate, either alone or with ADP, does not appear to give any degree of protection against inactivation. The difference in the protection achieved in the presence of ADP or ATP points to the role of Lys 15 in the coordination of the y-phosphate of ATP and that this interaction is necessary to achieve the conformational change in the enzyme required for catalysis.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biophysics, molecular biology.
Subjects: Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Price, Prof. Nicholas
Date of Award: 2002
Depositing User: Enlighten Team
Unique ID: glathesis:2002-71063
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 18 May 2021 11:42

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