Structural Biology

Aspartate aminotransferase gene consists of 1,182 bp nucleotide encodes for 393 amino acids was sequenced, cloned, expressed and purified to homogeneity. Enzyme exhibited maximum activity at pH 7 at 65 ºC. Mass spectrometry determined molecular mass of 42,562 Da and gel filtration indicated the protein exist as a dimeric form. Thermo stability experiment showed 100% stability of a protein at 65 ºC for 16 hours and half-life of 15 mins at 75 ºC. The thermal denaturation studies by CD spectroscopy showed no significant change in ellipticity of the helical structure of the protein below 70 ºC. Km and Vmax values towards aspartate were 1.61 mM and 97 µmol min-1 mg-1 and towards α-ketoglutarate were 2.5 mM and 50 µmol min-1 mg-1, respectively. Substrate specificity experiment indicated maximum activity with aspartate and its respective keto acid (α-ketoglutarate ) while exhibits 27% of activity with Tyr and 16% of its activity with Pro and Cys and no activity with Glu how ever in reverse reaction of Glutamate with its keto acid, oxaloacetate, 70% of activity was observed. Pyridoxal phosphate quantification exhibited 0.1 mole of PLP per mole of enzyme. Amino acid analysis showed high contents of charged and especially acidic residues of Aspartate and glutamate in structure of ASTSBS enzyme. Homology modeling determined the Dimeric structure of ASTSBS which contained high number of proline on a surface of each sub-units as compare to its mesophilic counter parts that is a reason for stability of a protein at high temperature.