Mapping the structural domains of E. coli carbamoyl phosphate synthetase using limited proteolysis.
The structural and functional domains of Escherichia coli carbamoyl phosphate synthetase (CPS) have been identified by limited proteolysis. Incubation of CPS with several proteases, including trypsin, chymotrypsin, subtilisin and endoproteinase Asp-N, under native conditions, causes a time-dependent loss of enzymatic activity and the generation of a common fragmentation pattern. Amino-terminal sequencing studies demonstrated that the initial cleavage event by trypsin occurred at the carboxy-terminal end of the large subunit. The ultimate fragments produced in most of the proteolysis studies, 35- and 45-kDa peptides, were derived from areas corresponding to the putative ATP binding regions. Substrate protection studies showed that the addition of ligands did not affect the final fragmentation pattern of the protein. However, ornithine and UMP were found to significantly reduce the rate of inactivation by inhibition of proteolytic cleavage. MgATP and IMP provided modest protection whereas bicarbonate and glutamine showed no overall effect on proteolysis. Limited proteolysis by endoproteinase Asp-N resulted in the production of a fragment (or multiple fragments) which contained enzymatic activity but had lost all regulation by the allosteric ligands, UMP and ornithine. The small subunit has been shown to be protected from proteolysis by the large subunit. Proteolysis of the isolated small subunit resulted in the generation of a stable 31-kDa species which contained 10% of the original glutaminase activity. These studies demonstrate that a portion of the C-terminal end of the large subunit can be excised without entirely destroying the ability of CPS to catalyze the formation of carbamoyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
Mareya
S M
SM
Department of Chemistry, Texas A & M University, College Station 77843, USA.
Raushel
F M
FM
D000595:Amino Acid Sequence; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry*; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000138:chemical synthesis; D010449:Peptide Mapping; D010710:Phosphates; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D009943:Organophosphorus Compounds; D010710:Phosphates; C015732:carboxy phosphate; D010450:Endopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)