title: Reconstitution of active catalytic trimer of aspartate transcarbamoylase from proteolytically cleaved polypeptide chains.
abstract: Treatment of the catalytic (C) trimer of Escherichia coli aspartate transcarbamoylase (ATCase) with alpha-chymotrypsin by a procedure similar to that used by Chan and Enns (1978, Can. J. Biochem. 56, 654-658) has been shown to yield an intact, active, proteolytically cleaved trimer containing polypeptide fragments of 26,000 and 8,000 MW. Vmax of the proteolytically cleaved trimer (CPC) is 75% that of the wild-type C trimer, whereas Km for aspartate and Kd for the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, are increased about 7- and 15-fold, respectively. CPC trimer is very stable to heat denaturation as shown by differential scanning microcalorimetry. Amino-terminal sequence analyses as well as results from electrospray ionization mass spectrometry indicate that the limited chymotryptic digestion involves the rupture of only a single peptide bond leading to the production of two fragments corresponding to residues 1-240 and 241-310. This cleavage site involving the bond between Tyr 240 and Ala 241 is in a surface loop known to be involved in intersubunit contacts between the upper and lower C trimers in ATCase when it is in the T conformation. Reconstituted holoenzyme comprising two CPC trimers and three wild-type regulatory (R) dimers was shown by enzyme assays to be devoid of the homotropic and heterotropic allosteric properties characteristic of wild-type ATCase. Moreover, sedimentation velocity experiments demonstrate that the holoenzyme reconstituted from CPC trimers is in the R conformation. These results indicate that the intact flexible loop containing Tyr 240 is essential for stabilizing the T conformation of ATCase. Following denaturation of the CPC trimer in 4.7 M urea and dilution of the solution, the separate proteolytic fragments re-associate to form active trimers in about 60% yield. How this refolding of the fragments, docking, and association to form trimers are achieved is not known.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley 94720.
authors.forename: V M
authors.initials: VM
authors.lastname: Powers
authors.forename: Y R
authors.initials: YR
authors.lastname: Yang
authors.forename: M J
authors.initials: MJ
authors.lastname: Fogli
authors.forename: H K
authors.initials: HK
authors.lastname: Schachman
mesh_terms: D000495:Allosteric Site; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives; D002918:Chymotrypsin; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000302:isolation & purification; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives; D011487:Protein Conformation; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D010446:Peptide Fragments; D001224:Aspartic Acid; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D002918:Chymotrypsin; D010746:Phosphonoacetic Acid