In Situ Behavior of Pyrimidine Pathway Enzymes in Saccharomyces cerevisiae 4. The Channeling of Carbamylphosphate to Aspartate Transcarbamylase and Its Partition in the Pyrimidine and Arginine Pathways

Penverne, Bernadette; Belkaïd, Mohamed; Hervé, Guy

doi:10.1006/abbi.1994.1089

Cited by 31 publications

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“…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).…”

Section: Discussionmentioning

confidence: 99%

“…The transfer of intermediates requires physical association of the coupled enzymes either as stable complexes such as E. coli tryptophan synthetase (8,9) or by transient contact as proposed for glycolytic enzymes (10). Partial channeling of carbamoyl phosphate has been reported in the pyrimidine biosynthetic complexes from yeast (11)(12)(13), Neurospora (14,15), and mammals (16 -21) and in the mammalian urea cycle enzymes (22,23).…”

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confidence: 99%

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

Purcarea

Evans

Hervé

1999

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Purcarea

Evans

Hervé

1999

Journal of Biological Chemistry

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“…M. jannaschii has circumvented the problem of dUTP accumulation by directly coupling dCTP deamination to the release of pyrophosphate using the MjDCD-DUT enzyme described here. This strategy of sequestering dUTP is similar to the use of multifunctional, substrate channeling enzyme complexes by eukaryotes to catalyze the initial steps in pyrimidine biosynthesis (30,31).…”

Section: Resultsmentioning

confidence: 99%

The Methanococcus jannaschiidCTP Deaminase Is a Bifunctional Deaminase and Diphosphatase

Graham

et al. 2003

Journal of Biological Chemistry

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Most bacteria produce the dUMP precursor for thymine nucleotide biosynthesis using two enzymes: a dCTP deaminase catalyzes the formation of dUTP and a dUTP diphosphatase catalyzes pyrophosphate release. Although these two hydrolytic enzymes appear to catalyze very different reactions, they are encoded by homologous genes. The hyperthermophilic archaeon Methanococcus jannaschii has two members of this gene family. One gene, at locus MJ1102, encodes a dUTP diphosphatase, which can scavenge deoxyuridine nucleotides that inhibit archaeal DNA polymerases. The second gene, at locus MJ0430, encodes a novel dCTP deaminase that releases dUMP, ammonia, and pyrophosphate. Therefore this enzyme can singly catalyze both steps in dUMP biosynthesis, precluding the formation of free, mutagenic dUTP. Besides differing from the previously characterized Salmonella typhimurium dCTP deaminase in its reaction products, this archaeal enzyme has a higher affinity for dCTP and its steady-state turnover is faster than the bacterial enzyme. Kinetic studies suggest: 1) the archaeal enzyme specifically recognizes dCTP; 2) dCTP deamination and dUTP diphosphatase activities occur independently at the same active site, and 3) both activities depend on Mg 2؉ . The bifunctional activity of this M. jannaschii enzyme illustrates the evolution of a suprafamily of related enzymes that catalyze mechanistically distinct reactions.Deoxyuridine nucleotides pose severe problems for cells. Although necessary precursors for thymine nucleotide biosynthesis, deoxyuridine nucleotides can interfere with DNA polymerase activity, either by inhibiting polymerases or by causing them to misincorporate 2Ј-deoxyuridine 5Ј-triphosphate (dUTP) in place of 2Ј-deoxythymidine 5Ј-triphosphate (dTTP) (1, 2). Ubiquitous DNA glycosylase enzymes remove uracil bases from DNA, which are otherwise formed by spontaneous cytosine deamination. The resulting apyrimidinic sites are cleaved by endonucleases, fragmenting cellular DNA (3).At temperatures of 70 -95°C, rate constants for spontaneous cytosine deamination (in denatured DNA or cytidine nucleotides) are several orders of magnitude higher than those measured at more moderate temperatures (4). Therefore hyperthermophilic archaea that grow at such temperatures counter the mutagenic effects of cytosine deamination with a variety of thermostable uracil-DNA glycosylase enzymes (5). As a consequence of having efficient base excision repair mechanisms, these organisms must avoid misincorporating deoxyuridine nucleotides into DNA during replication by using highly discriminatory DNA polymerases and by maintaining low dUTP levels in the cell (1, 6).Eukaryotes, some bacteria and some archaea use zinc-dependent cytidine or deoxycytidine deaminase enzymes to produce uridine or deoxyuridine nucleotides. Yet many bacteria, including Escherichia coli and Salmonella typhimurium, produce most of their 2Ј-deoxyuridine 5Ј-monophosphate (dUMP) from 2Ј-deoxycytidine 5Ј-triphosphate (dCTP) using two different enzymes. These bacteria use dCTP deamina...

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“…The partial channeling of CP has been suggested to occur in pyrimidine biosynthetic complexes from yeast (53,54), Neurospora (55,56), and mammals (57,58) as well as between enzymes of the mammalian urea cycle (43,59). In addition, evidence for CP channeling in Thermus ZO5 (14), P. abyssi (15), and P. furiosus (8) was obtained from kinetic experiments, and we provide evidence for the formation of an enzyme complex directing the channeling of CP in P. furiosus.…”

Section: Discussionmentioning

confidence: 99%

Metabolic Channeling of Carbamoyl Phosphate, a Thermolabile Intermediate

Massant¹,

Verstreken²,

Durbecq³

et al. 2002

Journal of Biological Chemistry

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Two different approaches provided evidence for a physical interaction between the carbamate kinase-like carbamoyl-phosphate synthetase (CKase) and ornithine carbamoyltransferase (OTCase) from the hyperthermophilic archaeon Pyrococcus furiosus. Affinity electrophoresis indicated that CKase and OTCase associate into a multienzyme cluster. Further evidence for a biologically significant interaction between CKase and OTCase was obtained by co-immunoprecipitation combined with formaldehyde cross-linking experiments. These experiments support the hypothesis that CKase and OTCase form an efficient channeling cluster for carbamoyl phosphate, an extremely thermolabile and potentially toxic metabolic intermediate. Therefore, by physically interacting with each other, CKase and OTCase prevent the thermodenaturation of carbamoyl phosphate in the aqueous cytoplasmic environment.The thermal profile of several Pyrococcus proteins has been studied intensively (1-4), but much less is known about the thermolabile substrates and products that these thermostable enzymes utilize (5,6). Most of these intermediate metabolites are identical to the ones found in mesophiles, yet many are extremely thermolabile, and in some cases, their degradation products are toxic to the cell.A case in point is carbamoyl phosphate (CP), 1 a precursor of the pyrimidine and arginine pathways. Ornithine carbamoyltransferase (OTCase; EC 2.1.3.3) carbamoylates ornithine to form citrulline, which is converted into arginine in two steps. Aspartate carbamoyltransferase (EC 2.1.3.2) catalyzes the condensation of CP and aspartate into carbamoyl aspartate in the committed step of pyrimidine biosynthesis. The decomposition of CP in aqueous solutions at high temperatures (7) leads to the accumulation of cyanate, a powerful and indiscriminate carbamoylating agent. At 100°C, where Pyrococcus furiosus reaches its highest growth rate, the half-life of CP is less than 2 s (8). Thus, because CP is both extremely labile at high temperatures and potentially toxic, it would appear to require metabolic protection in vivo.Several examples exist that show that enzymes belonging to the same or related biochemical pathways are organized into functional complexes. These enzyme clusters can pass on products very efficiently without accomplishing a diffusion equilibrium with the bulk phase, a phenomenon called channeling (9 -13). Relatively tight channeling could protect chemically labile intermediates and thus play a critical role in the physiology of thermophiles.Competition experiments between labeled CP produced by carbamate kinase-like carbamoyl-phosphate synthetase (CKase; EC 2.7.2.2) and unlabeled CP added to P. furiosus cell extracts showed a marked preference of OTCase for the utilization of CP synthesized by CKase (8). These findings suggest that CKase and OTCase associate to form a multienzyme complex able to channel this labile intermediate. Similar findings indicated the existence of channeling of CP to both aspartate carbamoyltransferase and OTCase in Thermus ZO5 (14)...

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In Situ Behavior of Pyrimidine Pathway Enzymes in Saccharomyces cerevisiae 4. The Channeling of Carbamylphosphate to Aspartate Transcarbamylase and Its Partition in the Pyrimidine and Arginine Pathways

Cited by 31 publications

References 0 publications

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

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

The Methanococcus jannaschiidCTP Deaminase Is a Bifunctional Deaminase and Diphosphatase

Metabolic Channeling of Carbamoyl Phosphate, a Thermolabile Intermediate

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