Divergent allosteric patterns verify the regulatory paradigm for aspartatetranscarbamylase

The native Escherichia coli aspartate transcarbamoylase (ATCase, E.C. 2.1.3 .2) provides a classic allosteric model for the feedback inhibition of a bi osynthetic pathway by its end products. Both E. coli and Erwinia herbicola possess ATCase holoenzymes which are dodecameric (2(c(3)):3(r(2))) with 311 amino acid residues per catalytic monomer and 153 and 154 amino acid resid ues per regulatory (r) monomer, respectively. While the quaternary structur es of the two enzymes are identical, the primary amino acid sequences have diverged by 14 % in the catalytic polypeptide and 20 % in the regulatory po lypeptide. The amino acids proposed to be directly involved in the active s ite and nucleotide binding site are strictly conserved between the two enzy mes; nonetheless, the two enzymes differ in their catalytic and regulatory characteristics. The E. coli enzyme has sigmoidal substrate binding with ac tivation by ATP, and inhibition by CTP, while the E. herbicola enzyme has a pparent first order kinetics at low substrate concentrations in the absence of allosteric ligands, no Am activation and only slight CTP inhibition. In an apparently important and highly conserved characteristic, CTP and UTP i mpose strong synergistic inhibition on both enzymes. The co-operative bindi ng of aspartate in the E. coli enzyme is correlated with a T-to-R conformat ional transition which appears to be greatly reduced in the E. herbicola en zyme, although the addition of inhibitory heterotropic ligands (CTP or CTP + UTP) reestablishes co-operative saturation kinetics. Hybrid holoenzymes a ssembled in vivo with catalytic subunits from E. herbicola and regulatory s ubunits from E. coli mimick the allosteric response of the native E. coli h oloenzyme and exhibit ATP activation. The reverse hybrid, regulatory subuni ts from E. herbicola and catalytic subunits from E. coli, exhibited no resp onse to ATP. The conserved structure and diverged functional characteristic s of the E. herbicola enzyme provides an opportunity for a new evaluation o f the common paradigm involving allosteric control of ATCase. (C) 1999 Acad emic Press.