Student Number : 0002483R -
MSc dissertation -
School of Molecular and Cell Biology -
Faculty of Science / Isothermal titration calorimetry was used to investigate the forces that drive ligand
binding to the active site of rGST M1-1. In an attempt to gain insight into the
recognition of non-substrate ligands by GSTs, this study also investigates interactions
between rGST M1-1 and ANS, a non-substrate ligand. At 25 °C, complex formation
between rGST M1-1 and GSH, GSO3
-, and S-hexylglutathione is characterised by a
monophasic binding isotherm with Kd values of 38.5 mM, 2.1 mM and 0.2 mM,
respectively. One molecule of each ligand is bound per monomer of rGST M1-1.
Binding of these ligands is enthalpically favourable and entropically unfavourable
with a resultant favourable Gibbs free energy, overall. The effects of temperature and
buffer ionisation on the energetics of binding were studied. The enthalpic and
entropic contributions for all three ligands exhibited temperature dependence over the
temperature range investigated (5-30 °C). The Gibbs free energy showed negligible
changes with increasing temperature due to enthalpy-entropy compensation. The
temperature dependence of the binding enthalpy yielded heat capacity changes of –
2.69 kJ/mol/K and –3.68 kJ/mol/K at 25 °C for GSH and S-hexylglutathione binding
and –1.86 kJ/mol/K overall for GSO3
-. The linear dependence of DH on temperature
for GSO3
- binding to rGST M1-1 suggests the formation of a more constrained
complex which limits the fluctuations in conformations of the mu-loop at the active
site. The non-linear dependence of DH on temperature for GSH and Shexylglutathione
binding to the enzyme suggests the formation of a complex that
samples different bound conformations due to the mobility of the mu-loop even after
ligand is bound. Calorimetric binding experiments in various buffer systems with
different ionisation enthalpies suggest that the binding of GSH to rGST M1-1 is
coupled to the deprotonation of the thiol of GSH while GSO3
- binding to rGST M1-1
is independent of the buffer ionisation. At 25 °C, the rGST M1-1#1;ANS association is
represented by a monophasic binding isotherm with one molecule of ANS bound per
monomer of rGST M1-1. The interaction is both enthalpically and entropically driven
with a Kd value of 27.2 mM representing moderate affinity. The effect of temperature
on the interaction was investigated over the temperature range of 5-30 °C. The linear
dependence of the binding enthalpy on temperature indicates that no significant
structural changes occur upon binding of ANS to the enzyme (DCp = -0.34 kJ/mol/K).
The change in heat capacity associated with the interaction can be attributed to the burial of the polar sulphonate group of ANS and the exposure of the anilino and
naphthyl rings to solvent as well as the possibility of weak electrostatic interactions
between ANS and residues at the active site. The effect of ethacrynic acid, GSH,
GSO3
- and S-hexylglutathione on the fluorescence of ANS was investigated in order
to obtain some idea as to the location of the ANS binding site on rGST M1-1. ANS
was displaced by GSO3
-, S-hexylglutathione and ethacrynic acid, while no
displacement occurred upon binding of GSH to the active site of rGST M1-1.
Displacement studies and molecular docking simulations indicate that ANS binds to
the H-site of rGST M1-1 and the possibility of a second binding site for the molecule
cannot be ruled out.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/1715 |
| Date | 14 November 2006 |
| Creators | Kinsley, Nichole Michelle |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 1244958 bytes, application/pdf |
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