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Reconstitution of active catalytic trimer of aspartate transcarbamoylase from proteolytically cleaved polypeptide chains
Author(s) -
Powers Vincent M.,
Yang Ying R.,
Fogli Michael J.,
Schachman H. K.
Publication year - 1993
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560020613
Subject(s) - aspartate carbamoyltransferase , trimer , chemistry , stereochemistry , protein subunit , allosteric regulation , crystallography , biochemistry , enzyme , dimer , organic chemistry , gene
Treatment of the catalytic (C) trimer of Escherichia coli aspartate transcarbamoylase (ATCase) with α ‐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. V max of the proteolytically cleaved trimer (C PC ) is 75% that of the wild‐type C trimer, whereas K m for aspartate and K d for the bisubstrate analog, N ‐(phosphonacetyl)‐l‐aspartate, are increased about 7‐ and 15‐fold, respectively. C PC 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 C PC 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 C PC 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 C PC 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.