Divergent regulation in pyrimidine biosynthesis : enzymatic characterization of hybrid enteric aspartate transcarbamoylases and their physiological consequences

Abstract

The enzyme aspartate transcarbamoylase, (ATCase, EC 2.1.3.2), from Escherichia coli is composed of six regulatory (r) and six catalytic (c) polypeptides with an architectural composition described as a 2(c(,3)):3(r(,2)) oligomer. The allosteric regulation of the enzyme from E. coli is mediated by conformational changes in the bonding at one or more of the intermolecular domains formed by the interaction of its constituent polypeptides. The naive enzyme from E. coli shows cooperative affinity for substrates and exhibits allosteric activation by ATP and inhibition by CTP. The ATCase from Serratia marcescens is regulated quite differently showing increased substrate requirements and heterotropic activation by both ATP and CTP. In order to examine the nature of the regulatory responses in these enzymes, we have produced intergeneric hybrid ATCases which possess unique domains of bonding. The ATCases of S. marcescens and E. coli exhibit distinct regulatory and kinetic characteristics and were dissociated into regulatory (r(,2)) and catalytic (c(,3)) subunits. Hybrid enzymes with catalytic subunits from one organism and regulatory subunits from the other were formed as demonstrated by the molecular weight, the reestablishment of catalytic restraint, and the recovery of cooperative, homotropic responses. The hybrids formed are of special importance since ATCase from E. coli is inhibited by CTP and activated by ATP while the enzyme from S. marcescens is activated by both nucleotides. Both hybrid enzymes bound ATP and showed a positive heterotropic response and both were inhibited by CTP. Thus the kinetic and regulatory responses of the hybrids formed were not solely influenced by the characteristics of either the catalytic or regulatory subunits used to form the hybrid enzymes. The regulatory responses of these enzymes must be a consequence of characteristics of both the regulatory and catalytic chains as they reestablish r:c bonding domains. The ATCase genes from S. marcescens and E. coli were cloned into E. coli mutants. When catalytic and regulatory genes were expressed in trans from separate replicons in the same cell, complete cytoplasmic assembly of hybrid ATCase from nascent polypeptides was achieved. These results indicated that in vivo assembly of ATCase does not require interactions of nascent catalytic and regulatory polypeptides while they are still associated with the ribosomes, but that assembly of the holoenzyme occurs in the cytoplasm from preformed subunits.