Domain structure of rat liver carbamoyl phosphate synthetase I.
Independently folded structural domains of rat liver carbamoyl phosphate synthetase I have been identified by partial proteolytic cleavage under nondenaturing conditions. The pattern of fragments produced was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal sequences of the fragments were determined by automated Edman degradation. Comparison of these fragment sequences with the sequence of the intact protein allowed alignment of the fragments. The hydrolysis of carbamoyl phosphate synthetase I (Mr 160,000) by either trypsin or elastase proceeded in two stages, with two alternative routes of degradation for elastase. The alignment of the final tryptic fragments from the NH2 terminus to the COOH terminus was: Mr 87,000 fragment-Mr 62,000 fragment-group of small peptides. The alignment of the final elastase fragments was: Mr 37,000 fragment-Mr 108,000 fragment-group of small peptides. The rates of cleavage were affected by the presence of the substrate ATP or the positive allosteric effector N-acetylglutamate; the preferred route of elastase cleavage was also affected. In addition to providing a map of the carbamoyl phosphate synthetase I domains and preliminary information on the interaction of substrates with these domains, the present studies provide further support for the proposal that domains serve as units of protein evolution since the 37-kDa fragment encompasses the region of the rat liver synthetase that is homologous to the 40-kDa subunit of the Escherichia coli synthetase.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism; D005971:Glutamates / Q000494:pharmacology; D007700:Kinetics; D008025:Ligases / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D014357:Trypsin / Q000378:metabolism
D005971:Glutamates; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D010196:Pancreatic Elastase; D014357:Trypsin; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C016195:N-acetylglutamic acid