The Smallest Carbamoyl-phosphate Synthetase

Abstract

Escherichia coli carbamoyl-phosphate synthetase (CPSase) is comprised of a 40-kDa glutaminase (GLN) and a 120-kDa synthetase (CPS) subunit. The CPS subunit consists of two homologous domains, CPS.A and CPS.B, which catalyze the two different ATP-dependent partial reactions involved in carbamoyl phosphate synthesis. Sequence similarities and controlled proteolysis experiments suggest that the CPS subdomains consist, in turn, of three subdomains, designated A1, A2, A3 and B1, B2, B3 for CPS.A and CPS.B, respectively. Previous studies of individually cloned CPS.A and CPS.B from E. coli and mammalian CPSase have shown that homologous dimers of either of these "half-molecules" could catalyze all three reactions involved in ammoniadependent carbamoyl phosphate synthesis. Four smaller recombinant proteins were made for this study as follows: 1) A1-A2 in which the A3 subdomain was deleted from CPS.A, 2) B1-B2 lacking subdomain B3 of CPS.B, 3) the A2 subdomain of CPS.A, and 4) the B2 subdomain of CPS.B. When associated with the GLN subunit, A1-A2 and B1-B2 had both glutamine-and ammonia-dependent CPSase activities comparable to the wild-type protein. In contrast, the 27-kDa A2 and B2 recombinant proteins, which represent only 17% of the mass of the parent protein, were unable to use glutamine as a nitrogen donor, but the ammonia-dependent activity was enhanced 14 -16-fold. The hyperactivity suggests that A2 and B2 are the catalytic subdomains and that A1 and B1 are attenuation domains which suppress the intrinsically high activity and are required for the physical association with the GLN subunit.Carbamoyl-phosphate synthetase (CPSase, 1 EC 6.3.5.5) catalyzes the formation of carbamoyl phosphate from bicarbonate, NH 3 , usually derived from glutamine, and ATP (1, 2). The structure of CPSases can be quite diverse. For example, the monofunctional Escherichia coli CPSase (3, 4) consists of a 40-kDa glutaminase (GLN) subunit and a 120-kDa synthetase (CPS) subunit, whereas in its mammalian counterpart (5-7), the GLN and CPS domains are fused and are part of CAD, a multifunctional protein that also has aspartate transcarbamoylase and dihydroorotase activities. Despite the differences in structural organization, the amino acid sequences are clearly similar (8 -21) suggesting that all of these molecules are comprised of homologous domains and subdomains with analogous functions.The isolated GLN subunit of E. coli CPSase (3, 4) and the separately cloned GLN domain of CAD (22) hydrolyze glutamine (Reaction 1) and transfer ammonia to the CPS domain.All of the other partial reactions (Reactions 2-4) occur on the CPS domain or subunit (3, 4). The determination of the amino acid sequence of CPSase from many different organisms (8 -21) revealed that the CPS domain of these molecules invariably consists of two highly homologous halves, designated CPS.A and CPS.B. The two ATP-dependent partial reactions (Reactions 2 and 4) occur at different sites, and there is now convincing evidence (23-26) that CPS.A catalyzes the activation of bic...

Channeling of Carbamoyl Phosphate to the Pyrimidine and Arginine Biosynthetic Pathways in the Deep Sea Hyperthermophilic Archaeon Pyrococcus abyssi

“…Channeling or molecular compartmentation may represent one such mechanism for sequestering carbamoyl phosphate and directing it from its site of synthesis to either ATCase or OTCase where it can be efficiently and rapidly utilized. There is precedent for partial channeling of pyrimidine biosynthetic intermediates in Saccharomyces cerevisiae (11)(12)(13), Neurospora crassa (14 -15), and mammals (16,17,38). Moreover, there is convincing evidence for carbamoyl phosphate channeling from CPSase I to OTCase in rat liver mitochondria (22,23) in Thermus aquaticus Z05 (39), a hyperthermophilic eubacteria, and Pyrococcus furiosus (45).…”

Cell Cycle-dependent Regulation of Pyrimidine Biosynthesis

“…Enzyme Assays-The glutamine-dependent CPSase assay has been described (39). The 1-ml assay mixture contained 100 g of protein, 100 mM Tris-HCl, pH 8.0, 100 mM KCl, 7.5% Me 2 SO, 2.5% glycerol, 1 mM dithiothreitol, 3.5 mM glutamine, 20.2 mM aspartate, 1.5 mM ATP, 3.5 mM MgCl 2 , and 5 mM sodium 14 C-labeled bicarbonate (1.6 ϫ 10 6 Ci/ mol).…”

Required Allosteric Effector Site for -Acetylglutamate on Carbamoyl-Phosphate Synthetase I

“…Refs. 24,25), and the remainder of the protein can function independently for ammoniadependent carbamoyl phosphate synthesis; however, there is a strong functional linkage between occupancy of the glutamine site on the GAT domain and occupancy of the ATP B site on the synthetase portion of CPSase, with greatly increased activity at each site resulting from occupancy of the other site (6,24,25).…”

“…(d) Diversification of both subdomains A-1 and A-2 occurred to yield CPSase I with subdomain A-1 involved in binding AGA and subdomain A-2 no longer retaining GAT activity. The utilization of AGA as a required allosteric activator for CPSase I, with linkage between occupancy of the AGA site and the ATP B site (22,30), is analogous to the strong functional linkage between occupancy of the glutamine site on the GAT moiety and occupancy of the ATP B site on the synthetase moiety in the CPSase II family (6,25). However, in the CPSase I case, the activator AGA interacts directly with the interaction subdomain A-1 and can directly mediate a functional response at the ATP B site, whereas in the CPSase II case, glutamine binds to subdomain A-2, and this occupancy must be communicated to the ATP B site via the interaction subdomain A-1.…”