title: Structure and function of aspartate transcarbamoylase studied using chymotrypsin as a probe.
abstract: Aspartate transcarbamoylase from Escherichia coli is composed of six catalytic (c) and six regulatory (r) polypeptides. We have studied the structure and function of this enzyme using chymotrypsin as a probe. The protease inactivates the isolated catalytic subunit (c3) but has not effects on the native enzyme (c6r6). Under identical conditions, the c3r6 complex is inactivated at a much slower rate than c3. The presence of the substrate analogue succinate together with carbamoyl phosphate reduces substantially the rate of inactivation. Extended exposure to chymotrypsin converts the catalytic subunit into a partially active derivative with a fourfold higher Michaelis constant. This derivative is indistinguishable from the unmodified catalytic subnit in gell electrophoresis under nondenaturing conditions. However, in the presence of sodium dodecyl sulfate, the major fragment in the electropherogram is smaller than that of the intact catalytic polypeptide. The results could be explained by postulating the presence of a chymotrypsin-sensitive peptide bond at or near the active site. Since X-ray crystallographic studies have indicated that the active sites are located in a central cavity, the resistance of the native enzyme towards inactivation may be due to the inability of chymotrypsin to enter this cavity.
mesh_terms: D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D002221:Carbamyl Phosphate / Q000494:pharmacology; D002918:Chymotrypsin / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013386:Succinates / Q000494:pharmacology
chemical_list: D046911:Macromolecular Substances; D013386:Succinates; D001224:Aspartic Acid; D002221:Carbamyl Phosphate; D001221:Aspartate Carbamoyltransferase; D002918:Chymotrypsin