Conversion of the Allosteric Regulatory Patterns of Aspartate Transcarbamoylase by Exchange of a Single β-Strand between Diverged Regulatory Chains

Aspartate transcarbamoylase (EC 2.1.3.2; ATCase)1 provides the first unique step of the pathway for de novo pyrimidine biosynthesis by catalyzing the condensation of L-aspartate with carbamoyl phosphate to form carbamoyl aspartate and phosphate, ultimately leading to the end products UTP and CTP (Jones et al., 1995; Reichard & Hanshoff, 1956). The regulation of this transcarbamoylation varies from substrate channeling in multienzyme complexes to allosteric control of independent enzymes composed of distinct catalytic and regulatory subunits in various biological systems. Multiple structures of the Escherchia coli (Ec) ATCase (2c3:3r2)2 have been characterized by X-ray crystallography [Gouaux et al., 1990; Stevens et al., 1990; Ke et al., 1988; Kosman et al., 1993; for review see Lipscomb (1994)], and the relationship between structure and function has been intensively investigated using single-site-directed mutagenesis [for review see Kantrowitz and Lipscomb (1990); Stevens et al., 1991]. The ATCase holoenzymes from Ec and other enteric bacteria are expressed from the pyrLBI operons. The pyrB and pyrI cistrons encode the catalytic and regulatory polypeptides, respectively. Three catalytic polypeptides associate to form a functional catalytic subunit (trimer, c3), and two regulatory polypeptides form a regulatory subunit (dimer, r2). The holoenzyme is composed of two catalytic subunits and three regulatory subunits (2c3:3r2). Each of the catalytic polypeptides is composed of a carbamoyl phosphate binding domain (CP domain) and an aspartate binding domain (Asp domain), while each regulatory polypeptide is folded into an allosteric effector binding domain (Allo domain) and a zinc binding domain (Zn domain). The active sites are found at the interfaces of the Asp and CP domains of the catalytic trimers, 60 A away from the nucleotide binding sites which are located adjacent to the five-stranded β-sheets of the Allo domains and connected to the Zn domains through the S5′ β-strand (Figure 1). Figure 1 (A, top) Molecular graphic representation of a CTP-liganded R-state holoenzyme of ATCase from Ec. The holoenzyme is composed of two catalytic trimers (one trimer is shown in blue while the second is located beneath this trimer and not shown here for the ... When substrate ligands bind to the native Ec ATCase, there is a conformational change from a less active “T-state” to the more active, open “R-state” (Schachman et al., 1988; Lipscomb, 1994). As the T → R transition occurs, the catalytic trimers move apart by 12 A, and the catalytic and regulatory subunits rotate 10° and 15° above their axes of symmetry, respectively (Krause et al., 1987). The holoenzyme exhibits both homotropic and heterotropic allosteric regulation, by which the positive cooperativity of binding of aspartate can be modulated (Gerhart & Schachman, 1968; Bethell et al., 1968). The nucleotide effectors competitively bind to the r2 dimers and either activate (ATP) or inhibit (CTP, CTP + UTP) the formation of carbamoyl aspartate (Gerhart & Pardee, 1962; Wild et al., 1989). The ATCase from Serratia marcescens (Sm) has the same dodecameric structure, 2c3:3r2, as the Ec enzyme and shares 85% and 76% amino acid identity within their catalytic and regulatory polypeptides, respectively (Beck et al., 1989; this study). The regulatory polypeptide of Sm ATCase has 154 amino acids, one more than the Ec; however, a direct comparison of the sequences of the Sm and Ec regulatory polypeptides indicates there are only 37 amino acid differences between the two. The most divergent regions are involved in the minor r1:c1 interface (r130’s region) and the Zn:Allo domain junction (r100’s region) (Beck et al., 1989; Wild et al., 1990; Wild & Wales, 1990; and Figure 2). Most of the residues involved in the nucleotide binding sites and all inter- and intrasubunit interfaces (r1:c1, r1:c4, r1:r6, and Allo:Zn) are conserved between the two enzymes (Gouaux et al., 1990; Beck et al., 1989). In spite of these apparent structural similarities, the two enzymes differ in both regulatory and catalytic characteristics. Most notably, CTP activates the catalytic activity of the Sm ATCase, and the CTP + UTP inhibitory synergism is lost. This allosteric pattern is contrary to the traditional logic of the Ec ATCase where CTP, the end product of the pathway, feedback inhibits 50–60% of the catalytic activity of the enzyme, and the combination of CTP and UTP inhibits 90–95%. A hybrid ATCase assembled in vivo with the catalytic subunits from Ec and the regulatory subunits from Sm (Cec:Rsm) exhibited heterotropic responses characteristics of the native Sm holoenzyme (ATP and CTP activation; Shanley et al., 1984; Beck et al., 1989). In contrast, the reverse hybrid enzyme (Csm:Rec), utilizing the Sm catalytic subunits and the Ec regulatory subunits, exhibited the heterotropic responses characteristic of Ec (Shanley et al., 1984; Beck et al., 1989). These hybrids, along with other interspecies hybrids, have verified the hypothesis that the allosteric responses of the ATCase holoenzyme are determined exclusively by the controlling regulatory subunits (Wild et al., 1990; Wild & Wales, 1990; Wales & Wild, 1991). Figure 2 Comparison of the amino acid sequence of the regulatory polypeptides between Ec and Sm ATCase. The standard single-letter abbreviations from the amino acids are used. The presence of a “.” indicates a shift in sequence to optimize the ... The structural similarity and the functional divergence of these two enzymes provide an opportunity to directly address the role of specific structural regions in allosteric regulation. In this study, a chimeric enzyme has been constructed in which the S5′ β-sheet of the Sm enzyme was replaced by that of the Ec enzyme. The resulting chimeric ATCase displays allosteric patterns similar to those of the native Ec enzyme (ATP activation, CTP inhibition, and CTP + UTP synergistic inhibition). These results suggest that the nonconserved residues r93–r97 are critical to defining the diverged allosteric function and that the nature of the allosteric response is directly modulated by this region.