Mechanism of the GSTT2 Enzyme

Glutathione transferases (GSTs) are enzymes that play a role in detoxication: removing harmful substances from a cell. They do this by linking a molecule, glutathione, to the harmful substance. GSTs allow this reaction to take place by facilitating the removal of a Hydrogen atom from a Sulfur atom in glutathione, forming a reactive thiolate anion. It is this anion that links to the substance to be removed. There are several classes of GSTs. The Theta and the Delta classes differ from the Alpha, Pi, Mu, and Sigma classes in that they lack the Tyr amino acid that stabilises the glutathione thioloate anione. Stabilisation is achieved through the Tyr hydroxyl group. Focusing on the Theta class enzyme GSTT2, the aim of this work is to investigate the role of a Ser amino acid in the active site that is also capable of donating a hydrogen bond through its hydroxyl group. To do this, high level ab initio calculations on model atomic systems of the active site were performed.

Principal Investigator

Gareth Chelvanayagam
Human Genetics


x06, x07

Facilities Used



Phil Board
Jack Flanagan
Human Genetics

William King
Molecular Biology

Michael Parker
Ian Potter Foundation Protein Crystallography Laboratory
St Vincent's Institute of Medical Research

RFCD Codes


Significant Achievements, Anticipated Outcomes and Future Work

It has been previously established that the role of the Tyr residue in the Alpha, Pi, Mu, and Sigma class GSTs is one of a surrogate water molecule, donating a proton through a conventional hydrogen bond to stabilise the thiolate. We have established that the hydrogen bond strength of the interaction between the GSTT2 Ser 11 and the glutathione thiolate is much weaker than in classes with an active site Tyr. Further, we have discovered second sphere interactions that, depending on the amino acids involved, markedly influence the first sphere hydrogen bond. In GSTT2, the interaction of Ser 14 with Ser 11 significantly increases the energy of the bond.

Based on these studies it can be hypothesised that by altering the position of the first sphere interaction, the thiolate could be oriented differently, thereby facilitating catalysis or transition state stabilisation of particular reactions.


Computational Techniques Used

This study was performed using the Gaussian 98 Revision A.6 (Gaussian Inc.) package, running on the ANU Supercomping Facility SGI Power Challenge. Atomic coordinates of the crystal structure of the GSTT2 enzyme were obtained from the authors. Based on this structure, model systems of the Ser:thiolate first sphere interaction were derived. The Ser residue was modeled as methanlo, whereas the thiol of the glutathione was modeled as methanethiol. Different orientations of the hydrogen bond donor systems were considered in both a neutral and anionic state. Similar second sphere interactions were considered involving Ser 14:Ser 11:thiolate and Cys 14:Ser 11:thiolate. Calculations were performed using RHF and MP2 levels of theory with different basis sets.


Publications, Awards and External Funding

External Funding:

This work was supported by the Ramaciotti Foundation, the ANU and the ARC.


J.U. Flanagan, W. King, M.W. Parker, P.G. Board and G. Chelvanayagam, Ab Initio Calculations on Hidden Modulators of Theta Class Glutathione Transferase Activity, Proteins: Structure, Function, and Genetics 39, 2000, 235-243.