Effect of growth temperature on folding of carbamoylphosphate synthetases of Salmonella typhimurium and a cold-sensitive derivative.
The properties of homogeneous preparations of carbamoylphosphate synthetase (CPSase) from wild-type Salmonella typhimurium and a cold-sensitive derivative grown at different growth temperatures were examined. For the cold-sensitive mutant, the affinity for glutamine of the form of CPSase synthesized at 20 degrees C was lower than that of the form of the enzyme synthesized at 37 degrees C, regardless of the assay temperature. Thus, the cold sensitivity of the mutant reflects an effect of temperature on the synthesis of the enzyme rather than the activity of the folded enzyme. The two forms also differed in sensitivities to polyclonal antibodies as well as denaturational enthalpies. The combined results support the hypothesis that carAB mutations conferring cold sensitivity identify amino acid residues that are critical in the folding of CPSase. Quite unexpectedly, certain kinetic properties of cloned parent CPSase were also dependent on the growth temperature, although to a much lesser extent than those of the cold-sensitive mutant. The specific activity of wild-type CPSase synthesized at 15 degrees C was 60% of that synthesized at 37 degrees C. Further, CPSase synthesized at 15 degrees C was less thermostable than the enzyme synthesized at 37 degrees C; the difference in stability (delta G) is estimated to be 4,500 cal mol-1. Thus, variation of temperature within the physiological range for growth influences the folding and consequently the properties of CPSase from wild-type S. typhimurium.
Laboratory for Microbial and Biochemical Sciences, Georgia State University, Atlanta 30302-4010.
B D
BD
Han
W G
WG
Nolan
H P
HP
Hopkins
R T
RT
Jones
J L
JL
Ingraham
A T
AT
Abdelal
D000643:Ammonium Chloride / Q000378:metabolism; D002151:Calorimetry; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000302:isolation & purification / Q000378:metabolism*; D003080:Cold Temperature; D007700:Kinetics; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology / Q000254:growth & development*; D013696:Temperature
D000643:Ammonium Chloride; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)