CLONING AND CHARACTERIZATION OF THE ARGININE-SPECIFIC CARBAMOYL-PHOSPHATE SYNTHETASE FROM BACILLUS-STEAROTHERMOPHILUS

Bacillus stearothermophilus contains two carbamoyl-phosphate synthetas es (CPS), one specific for pyrimidine biosynthesis and the other for a rginine biosynthesis. The pyrimidine specific CPS is repressed by exog enous pyrimidines, and its activity is inhibited by UMP and activated by 5-phospho-alpha-D-ribosyl diphosphate. The arginine-specific CPS is similarly repressed by exogenous arginine but its activity is not sen sitive to these or other potential effectors. Each of the two enzymes consist of two unequal subunits, as is the case for other microbial CP S; however, the lame subunit for the arginine-specific CPS is smaller than that for the pyrimidine-specific enzyme. Comparison of the derive d amino acid sequence for the cloned large subunit of the arginine-spe cific CPS with those for subunits from pyrimidine-sensitive CP showed significant similarity throughout the polypeptides except at the carbo xy terminus, which was identified by other laboratories to contain the binding site for the pyrimidine effector. Unlike the results previous ly reported for CPS from an enteric mesophile, the kinetic propel ties of the arginine-specific CPS were not affected by growth of B. stearo thermophilus at temperatures near the minimal growth temperature. Furt hermore, calorimetric studies showed that the thermal stability of clo ned CPS was identical regardless of the growth temperature of B. stear othermophilus between 42 degrees C and 63 degrees C. The thermal stabi lity of cloned CPS was not affected by expression at 37 degrees C in B acillus subtilis or Escherichia coli. In contrast, the thermal stabili ties for CPS and other proteins were higher in extracts of cells grown al higher temperatures. These results indicate that cellular factors, probably chaperonins, are necessary for thermal stability of proteins at and below the optimal temperature for this thermophile.