(1) Bioinformatics Centre, University of Pune, Ganeshkhind,
Pune, India.
(2) Interdisciplinary Centre for Biotechnology Research,
University of Florida, Gainsville, FL 32610
An approach based on following four main steps has been developed
to identify active site of an enzyme. These are :
1. Locating the invariant regions in a family of proteins from different
biological sources;
2. predicting the three dimensional structure of such region using Molecular
modelling approach;
3. docking of substrate/competitive inhibitor onto this modelled molecule
4. confirmation of the active site through site-directed mutagenesis
studies in laboratory.
This method was applied to predict the aspartate binding site of the enzyme
asparagine synthetase.
Asparagine synthetase is a key enzyme which increases the levels of asparagine in leukamic patients and thus specific inhibitor of this enzyme can help in treatment of such patients. Little physico-chemical or kinetic studies are available for this enzyme. Sequences of Asparagine synthetase from 6 different sources are aligned and invariant regions are identified. A threonine-rich invariant region around amino acids 316-332 is postulated to be the aspartate-binding site. Therefore molecular modelling and simulation of a 12-mer peptide soaked in water were carried out using the Biosym/MSI software InsightII and Discover. For the simulations, simulated annealing approach was used. The global minimum free energy conformation was found to be folded; having a cavity in which the aspartate molecule was docked. The docking interaction energy was calculated.
In the laboratory, several single point and double mutations were carried out in the in this region of the protien and Asparagine synthetase of E.coli It was found that the binding of cofactors glutamine and ATP does not get affected due to any mutations, though the aspartate binding is drastically affected in some of the mutated peptides. This indicates that the local conformation is altered due to mutations which was also observed in the molecular modelling studies of the 12mer peptide.
This approach, we feel is a general one and can be used in identifying active sites/binding sites on enzymes/proteins. Details of the approach and results will be discussed.