IN-VIVO FORMATION OF ALLOSTERIC ASPARTATE TRANSCARBAMOYLASE CONTAINING CIRCULARLY PERMUTED CATALYTIC POLYPEPTIDE-CHAINS - IMPLICATIONS FOR PROTEIN-FOLDING AND ASSEMBLY

Because the N- and C-terminal amino acids of the catalytic (c) polypep tide chains of Escherichia coli aspartate transcarbamoylase (ATCase) a re in close proximity to each other, it has been possible to form in v ivo five different active ATCase variants in which the terminal region s of the wild-type c chains are linked in a continuous polypeptide cha in and new termini are introduced elsewhere in either of the two struc tural domains of the c chain. These circularly permuted (cp) chains we re produced by constructing tandem pyrB genes, which encode the c chai n of ATCase, followed by application of PCR, Chains expressed in this way assemble efficiently in vivo to form active, stable ATCase variant s, Three such variants have been purified and shown to have the kineti c and physical properties characteristic of wild-type ATCase composed of two catalytic (C) trimers and three regulatory(R) dimers. The value s of V-max, for (cp)ATCase(122), (cp)ATCase(222), and (cp)ATCase(281) ranged from 16-21 mu mol carbamoylaspartate per mu g per h, compared w ith 15 for wild-type ATCase, and the values for K-0.5 for the variants were 4-17 mM aspartate, whereas wild-type ATCase exhibited a value of 6 mM. Hill coefficients for the three variants varied from 1.8 to 2.1 , compared with 1.4 for the wild-type enzyme. As observed with wild-ty pe ATCase, ATP activated the variants containing the circularly permut ed chains, as shown by the lowering of K-0.5 for aspartate and a decre ase in the Hill coefficient (n(H)) In contrast, CTP caused both an inc rease in K-0.5 and n(H) for the variants, just as observed with wild-t ype ATCase. Thus, the enzyme containing the permuted chains with widel y diverse N- and C-termini exhibited the homotropic and heterotropic e ffects characteristic of wild-type ATCase. The decrease in the sedimen tation coefficient of the variants caused by the binding of the bisubs trate ligand N-(phosphonacetyl)-L-aspartate (PALA) was also virtually identical to that obtained with wild-type ATCase, thereby indicating t hat these altered ATCase molecules undergo the analogous ligand-promot ed allosteric transition from the taut (T) state to the relaxed (R) co nformation. These ATCase molecules with new N- and C-termini widely di spersed throughout the c chains are valuable models for studying in vi vo and in vitro folding of polypeptide chains.