STABILITY OF ASPARTATE-AMINOTRANSFERASE FROM SULFOLOBUS-SOLFATARICUS

Aspartate aminotransferase from Sulfolobus solfataricus (SsAspAT) is a n extremely thermophilic and thermostable dimeric enzyme which retains its structure and reaches maximal activity at 100 degrees C, The stru ctural stability of this protein was investigated by coupling isotherm ally and thermally induced denaturation studies to molecular modeling, Gel filtration analysis indicated that SsAspAT unfolds with an N-2 re versible arrow 2D mechanism, In the molecular model, a cluster of hydr ophobic residues was shown at the interface between the subunits of Ss AspAT and suggested this cluster as a structural feature stabilizing t he enzyme quaternary structure, At 25 degrees C, SsAspAT is less resis tant to guanidinium chloride-induced denaturation than the cytosolic a spartate aminotransferase from pig heart (cpAspAT), which was chosen a s a mesophilic counterpart in the thermodynamic analysis since it shar es with SsAspAT the two-state unfolding mechanism, Therefore, in the c ase of aspartate aminotransferases, thermal stability does not correla te with the stability against chemical denaturants. Isothermal denatur ation curves at 25 degrees C and melting profiles recorded in the pres ence of guanidinium chloride showed that the Delta G degrees(H2O) at 2 5 degrees C of SsAspAT exceeds that of cpAspAT by roughly 15 kJ/mol; t he parameter bn, related to the number of binding sites for the denatu rant differentially exposed in unfolded and folded states, is higher f or SsAspAT than for cpAspAT; and Delta C-p is lower for the thermophil ic enzyme than for the mesophilic one by 8 kJ/K.mol. These results are indicative of a less hydrophobic core for SsAspAT than cpAspAT, In ag reement with this, the molecular model predicts that some charged side chains are buried in SsAspAT and interact to form an H-bond/ion-pair network.