Student Number : 0002482E -
MSc dissertation -
School of Molecular and Cell Biology -
Faculty of Science / Proteins in the glutathione transferase family share a common fold. The close packing of
secondary structures in the thioredoxin fold in domain 1 forms a compact hydrophobic
core. This fold has a bababba topology and most proteins/domains with this fold have a
topologically conserved isoleucine residue at the N-terminus of a-helix 3. Class Alpha
glutathione transferases are one of 12 classes within the glutathione transferase family.
To investigate the role of the conserved isoleucine residue in the structure, function and
stability of glutathione transferases, homodimeric human glutathione transferase A1-1
(hGST A1-1) was used as a representative of the GST family. Ile71 was replaced with
valine and the properties of I71V hGST A1-1 were compared with those of wildtype
hGST A1-1. The spectral properties monitored using far-UV CD and tryptophan
fluorescence indicated little change in secondary or tertiary structure confirming the
absence of any gross structural changes in hGST A1-1 due to the incorporation of the
mutation. Both wildtype and mutant dimeric proteins were determined to have a
monomeric molecular mass of 26 kDa. The specific activity of I71V hGST A1-1 (130
mmol/min/mg) was three times that of wildtype hGST A1-1 (48 mmol/min/mg). I71V
hGST A1-1 showed increased kinetic parameters compared to wildtype with a 10-fold
increase in kcat/Km for CDNB. The increase in Km of I71V hGST A1-1 suggests the
mutation had a negative effect on substrate binding. The DDG for transition state
stabilisation was –5.82 kJ/mol which suggest the I71V mutation helps stabilise the
transition state of the SNAR reaction involving the conjugation of reduced glutathione
(GSH) to 1-chloro-2,4-dinitrobenzene (CDNB). A 2-fold increase in the IC50 value for
I71V hGST A1-1 (11.3 mM) compared to wildtype (5.4 mM) suggests that the most
noticeable change due to the mutation occurs at the H-site of the active site.
Conformational stability studies were performed to determine the contribution of Ile71 to
protein stability. The non-superimposability of I71V hGST A1-1 unfolding curves and
the decreased m-value suggest the formation of an intermediate state. The conformational
stability of I71V hGST A1-1 (16.5 kcal/mol) was reduced when compared to that of the
wildtype (23 kcal/mol). ITC was used to dissect the binding energetics of Shexylglutathione
to wildtype and I71V hGSTA1-1. The ligand binds 5-fold more tightly
to wildtype hGST A1-1 (0.07 mM) than I71V hGST A1-1 (0.37 mM). The I71V mutant displays a larger negative DCp than wildtype hGST A1-1 (DDCp = -0.41 kJ/mol/K). This
indicates that a larger solvent-exposed hydrophobic surface area is buried for I71V hGST
A1-1 than for wildtype hGST A1-1 upon the binding of S-hexylglutathione. Overall the
results suggest that Ile71 conservation is for the stability of the protein as well as playing
a pivotal indirect role in catalysis and substrate binding.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/1726 |
| Date | 15 November 2006 |
| Creators | Fisher, Loren Tichauer |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 904281 bytes, application/pdf, application/pdf |
Page generated in 0.002 seconds