Flexibility of the GSTT2-2 Enzyme
Principal Investigator
Gareth Chelvanayagam
Division of Molecular Medicine,John Curtin School of Medical Research
Co-Investigators
Jack Flanagan
Philip Board
Division of Molecular Medicine, John Curtin School of Medical Research
Projects
x07 - PC, VPP
The glutathione transferases (GSTs) are a
superfamily of enzymes responsible for the
conjugation of the tripeptide GSH to generally toxic second substrates capable
of causing damage to other cellular macromolecules such as DNA. This renders
the toxic substrates more hydrophillic and consequently, less likely to
cause cellular damage. Furthermore, the conjugation of GSH provides the
cell with a metabolic handle through which it can proccess these toxins
and finally transport them from the cell. The GSTs catalyse the conjugation
of GSH by reducing the pKa of the cysteinyl thiol moiety of GSH allowing
it to form the thiolate anion which it can then stabilize. Stabilization
of the thiolate is carried out by a conserved N terminal domain hydroxyl
group, however the donor residue for this group is not conserved. Of the
four classes that have been the subject of most mechanistic studies, three
classes (Alpha, Mu and Pi) utilize a Tyr hydroxyl, whereas the Theta class
enzymes utilize a Ser hydroxyl.
The flexibility of the GSTT2-2 enzyme is of interest as the x-ray crystal structure of this enzyme did not provide any physical clues as to how substrates entered the active site despite the fact that the crystals were catalytically active. Experimental results have pointed to a region which potentially causes increased flexibility and faster substrate and product flux, indicating that this is also a very important feature of the enzyme's mechanistic study.
What are the results to date and the future work?
So far only preliminary work has begun on this project and most of our efforts have been directed towards our related project on the mechanism of the GSTT2 enzyme. Nonetheless, several design parameters of the system have been established and calculations will commence shortly.
Insights from this work have assisted us in the construction of an homology model for the related GSTT1 enzyme, including the identification of amino acids likely to be involved in the reaction mechanism.