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Title: Reconstitution of active catalytic trimer of aspartate transcarbamoylase from proteolytically cleaved polypeptide chains.

Authors: Powers, V M; Yang, Y R; Fogli, M J; Schachman, H K

Published In Protein Sci, (1993 Jun)

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.

PubMed ID: 8318885 Exiting the NIEHS site

MeSH Terms: Allosteric Site; Aspartate Carbamoyltransferase/chemistry; Aspartate Carbamoyltransferase/isolation & purification*; Aspartate Carbamoyltransferase/metabolism; Aspartic Acid/analogs & derivatives; Chymotrypsin; Enzyme Stability; Escherichia coli/enzymology; Kinetics; Models, Molecular; Peptide Fragments/isolation & purification; Phosphonoacetic Acid/analogs & derivatives; Protein Conformation; Substrate Specificity; X-Ray Diffraction

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