Structure and stability studies of shikimate kinase

[摘要] This thesis describes a structural and functional study of shikimate kinase, the enzyme which catalyses the fifth step in the biosynthetic shikimate pathway in which shikimate is converted to shikimate-3 -phosphate. Mutagenesis studies were undertaken to discover more about the roles of the assumed catalytic residues in particular the single lysine (K15) within the active site and the neighbouring cysteine (C13). The cysteine remote from the active site (C162) was also mutated to prevent intermolecular disulphide formation which had previously been observed during crystallographic studies. Biophysical techniques including circular dichroism (CD), fluorescence and microcalorimetry were used to monitor the conformational changes of shikimate kinase accompanying ligand binding and to measure ligand binding. The single tryptophan (W54) was found to be a very useful fluorophore for monitoring conformational changes. Calculated Kd values from this compared well with the Km values measured by steady state kinetics. The dissociation constant (Kd) for shikimate was 300 muM and for ATP 700 muM. When monitored by fluorescence the binding of the ATP to the enzyme showed a degree of co-operativity. The data did not fit a standard Michaelis Menten binding curve but gave a sigmoidal plot with a Hill co-efficient of approximately 2. The binding of ADP gave a Hill co-efficient of approximately 1.5. Fluorescence quenching studies using the quenching agents of succinimide and sodium iodide allowed the calculation of Stern-Volmer constants. The results provided information about the environment of the Trp-54. ATP binding to the enzyme was also measured by Isothermal titration calorimetry (ITC) This gave Kd values of only 25-40 muM whereas apparent steady state kinetics gave a Km value of 700 muM. This discrepancy has not been explained but it may reflect the existence of a second ATP binding site. Thermal stability was measured using differential scanning calorimetry (DSC) which was analysed to give the apparent melting temperature (Tm). Ligand binding (ATP) was found to stabilize the structure of shikimate kinase illustrated by an increase in the Tm from 39 to 47°C. However the shikimic acid provides a lower degree of stabilisation. The mutant K15M had a higher Tm than the wild type enzyme; this may be due to the formation of an extra ion pair in the structure, formed between the side chains of Asp 32 and Arg 11. Thermal stability was also measured by using CD to monitor the loss of secondary structure at 222nm. Denaturation and refolding studies were carried out using guanidine hydrochloride (GdnHCl) as the denaturant. The concentration of GdnHCl needed to bring about 50% of the total change of the native structure in both secondary, measuring the change in CD, and tertiary structure measuring the changes in fluorescence was 1.25M. This was observed as a single transition. Total loss of structure was observed at concentrations of 4M and above. Refolding of the enzyme was monitored by both fluorescence and CD using both manual mixing and stopped flow techniques. The data indicated that refolding took place in a number of distinctive stages. One of the intermediates appeared to have a high affinity for the fluorescent probe 1-anilino-8-napthalenesulphonate (ANS). An outline model for the refolding of the enzyme could be proposed.

[发布机构] University:University of Glasgow

[关键词] Molecular biology