RECONSTITUTION OF ACTIVE CATALYTIC TRIMER OF ASPARTATE TRANSCARBAMOYLASE FROM PROTEOLYTICALLY CLEAVED POLYPEPTIDE-CHAINS

Treatment of the catalytic (C) trimer of Escherichia coli aspartate tr anscarbamoylase (ATCase) with alpha-chymotrypsin by a procedure simila r to that used by Chan and Enns (1978, Can. J. Biochem. 56, 654-658) h as been shown to yield an intact, active, proteolytically cleaved trim er 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-typ e C trimer, whereas K(m) for aspartate and K(d) for the bisubstrate an alog, N-(phosphonacetyl)-L-aspartate, are increased about 7- and 15-fo ld, respectively. C(PC) trimer is very stable to heat denaturation as shown by differential scanning microcalorimetry. Amino-terminal sequen ce analyses as well as results from electrospray ionization mass spect rometry 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 lo op known to be involved in intersubunit contacts between the upper and lower C trimers in ATCase when it is in the T conformation. Reconstit uted holoenzyme comprising two C(PC) trimers and three wild-type regul atory (R) dimers was shown by enzyme assays to be devoid of the homotr opic and heterotropic allosteric properties characteristic of wild-typ e ATCase. Moreover, sedimentation velocity experiments demonstrate tha t the holoenzyme reconstituted from C(PC) trimers is in the R conforma tion. These results indicate that the intact flexible loop containing Tyr 240 is essential for stabilizing the T conformation of ATCase. Fol lowing denaturation of the C(PC) trimer in 4.7 M urea and dilution of the solution, the separate proteolytic fragments reassociate to form a ctive trimers in about 60% yield. How this refolding of the fragments, docking, and association to form trimers are achieved is not known.