10/8/15, 4:28 PM
`
`The Online Metabolic and Molecular Bases of Inherited Disease >
`
`85: Urea Cycle Enzymes
`
`Saul W. Brusilow; Arthur L. Horwich
`
`Abstract
`The urea cycle, which consists of a series of five biochemical reactions, has two roles. In order to prevent the accumulation of toxic nitrogenous
`compounds, the urea cycle incorporates nitrogen not used for net biosynthetic purposes into urea, which serves as the waste nitrogen product in
`mammals. The urea cycle also contains several of the biochemical reactions required for the de novo synthesis of arginine.
`Urea cycle disorders are characterized by the triad of hyperammonemia, encephalopathy, and respiratory alkalosis (the earliest objective evidence of
`encephalopathy). Five well-documented diseases (each with considerable genetic and phenotypic variability) have been described, each representing a
`defect in the biosynthesis of one of the normally expressed enzymes of the urea cycle. Four of these five diseases—deficiencies of carbamyl phosphate
`synthetase (CPS) (OMIM 237300), ornithine transcarbamylase (OTC) (OMIM 311250), argininosuccinic acid synthetase (AS) (OMIM 215700), and
`argininosuccinate lyase (AL) (OMIM 207900)—are characterized by signs and symptoms induced by the accumulation of precursors of urea, principally
`ammonium and glutamine. The most dramatic clinical presentation of these four diseases occurs in full-term infants with no obstetric risk factors who
`appear normal for 24 to 48 hours and then exhibit progressive lethargy, hypothermia, and apnea all related to very high plasma ammonium levels.
`Milder forms of these diseases occur; they may present with signs of encephalopathy at any age from infancy to adulthood. The most common of these
`late-onset diseases occurs in female carriers of a mutation at the OTC locus of one of their X chromosomes. The late-onset cases present with respiratory
`alkalosis and episodic mental status changes progressing, if not emergently treated, to cerebral edema, brainstem compression, and death. The acute
`encephalopathy is characterized by brain edema and swollen astrocytes, the cause of which is attributed to intraglial accumulation of glutamine resulting
`in osmotic shifts of water into the cell. Axons, dendrites, synapses, and oligodendroglia are normal. A fifth disease, arginase deficiency (OMIM 107830),
`is characterized by a clinical picture consisting of progressive spastic quadriplegia and mental retardation; symptomatic hyperammonemia, which can be
`life-threatening, occurs neither as severely or as commonly as in the other four diseases. Apart from OTC deficiency, which is inherited as an X-linked
`disorder, the other four diseases are inherited as autosomal-recessive traits. Carrier status of OTC mutations in women is determined by pedigree analysis
`and molecular methods. For fetuses at risk, antenatal diagnosis is available by a number of methods, particular to each disease, including enzyme analysis
`of fibroblasts cultured from aminocytes, as well as molecular (DNA) methods.
`Molecular genetic analysis of the urea cycle enzymes has addressed their structure and expression and has permitted DNA-based diagnosis of
`deficiency, in many cases by direct analysis of mutations. Using the cloned complementary DNA as probes, expression in liver of RNA for all the
`enzymes has been observed to be increased severalfold by starvation. RNA coding for the 160-kDa subunit of the CPS I homodimer is detected almost
`exclusively in the liver and translates a precursor protein representing the product of fusion of two ancestral prokaryotic subunits, joined with an N-
`terminal mitochondrial targeting sequence. Few mutations have been identified in this large coding sequence in affected pedigrees so far, but a restriction
`fragment-length polymorphism (RFLP) in the human CPS locus is useful in prenatal diagnosis of deficiency. OTC is also expressed principally in the
`liver, and its subunit is also translated as a precursor, comprising an N-terminal mitochondrial targeting sequence that functions via an α-helical structure
`and net positive charge, joined with a mature portion that resembles prokaryotic transcarbamylases. Mitochondrial import requires the action of a variety
`of components in the cytosol to maintain an import-competent conformation, in the outer mitochondrial membrane for recognition of the precursor, in
`both outer and inner membrane for protein translocation, and in the matrix for proteolytic processing and folding to the active conformation. Gene
`deletions have been observed in approximately 15 percent of affected males. More than 100 different single base substitutions have been identified,
`producing amino acid substitution in many cases, involving either of the two domains of the OTC subunit. In other cases, splicing is affected, either
`destabilizing the messenger RNA (mRNA) or frameshifting the subunit. Prenatal diagnosis can be offered to most women who are established as
`heterozygous carriers by pedigree analysis, allopurinol testing, or DNA analysis, using direct DNA analysis of fetal DNA where the mutation is known,
`or using RFLPs. Recombinant OTC retroviruses have transduced cultured hepatocytes of mice with inherited OTC deficiency, and recombinant OTC
`adenoviruses have been injected into newborn mutant animals with evidence of rescue of deficiency. These gene transfer experiments aim toward
`achieving stable long-term OTC expression. Argininosuccinate synthetase (AS) is programmed from a single locus, but a large number of homologous
`processed pseudogenes are localized throughout the genome. Expression of AS mRNA has been studied in cultured cells, where the level of mRNA is
`greatly increased in response to canavanine treatment and repressed by the presence of arginine. The AS coding sequence has been successfully
`transferred into both cultured cells and mouse bone marrow cells as an approach to AS deficiency of supplying enzyme activity outside the liver. Analysis
`of AS mutations reveals considerable heterogeneity in the position of mutation, with most composed of codon substitutions that produce unstable protein
`products. Where direct mutation analysis is not possible, a number of polymorphisms at the AS locus enable linkage study of affected pedigrees. Human
`AL is similar to avian δ-crystallins, in which a virtually identical protein is apparently used as a structural component. Analysis of AL mutants also
`reveals considerable heterogeneity. Arginase in human liver and red cells is a cytosolic enzyme distinct from a second mitochondrial-localized enzyme.
`Deficient patients have shown heterogeneity in the site of mutation. Two RFLPs at the locus have been identified.
`Treatment requires restriction of dietary protein intake and activation of other pathways of waste nitrogen synthesis and excretion. For patients
`deficient in CPS, OTC, and AS, treatment with sodium phenylbutyrate activates the synthesis of phenylacetylglutamine, which has a dual effect. By
`providing a new vehicle for waste nitrogen excretion, which suppresses residual urea synthesis in the late-onset group, a reserve urea synthetic capacity is
`
`about:blank
`
`Page 1 of 89
`
`Page 1 of 89
`
`Horizon Exhibit 2033
`Par v. Horizon
`IPR2017-01767
`
`

`

`10/8/15, 4:28 PM
`
`generated that may support nitrogen homeostasis when required. In patients deficient in AS and argininosuccinase, supplementation of the diet with
`arginine promotes the synthesis of citrulline in the former and argininosuccinate in the latter, both of which serve as waste nitrogen products.
`Outcome of treatment of neonatal-onset disease has been disappointing. Even those neonates treated prospectively prior to the onset of hyperammonemia
`are at high risk for neurologic deficits. Parents should be realistically counseled as to the likely outcome if the infant is rescued. Treatment of late-onset
`disease appears to preserve the neurologic status found at the start of therapy.
`Introduction
`The urea cycle serves two purposes: (1) it contains, in part, the biochemical reactions required for the de novo biosynthesis and degradation of arginine,
`and (2) it incorporates nitrogen atoms not retained for net biosynthetic purposes into urea, which serves as a waste nitrogen product. Campbell's review1
`of the comparative biochemistry of nitrogen metabolism describes other waste nitrogen products (ammonium and purines) found in other animals.
`It also has been proposed2 that the urea cycle plays an important role in the disposal of bicarbonate and hence on pH homeostasis. A number of
`arguments against this view have been offered.3 Perhaps the strongest case against this function of the urea cycle can be found in patients with complete,
`or nearly so, defects in one of the enzymes of the urea cycle; apart from respiratory alkalosis related to the stimulatory effect of ammonium on
`respiration, these patients have little evidence of a disorder of pH homeostasis. For example, 28 ornithine transcarbamylase (OTC)-deficient neonates
`who presented with hyperammonemia had a respiratory alkalosis as manifested by the following blood gases: pH 7.5; pCO2, 24 torr; HCO3, 19.3 mM.4
`As shown in Fig. 85-1, a respiratory alkalosis develops very early in the course of untreated hyperammonemia in an OTC-deficient neonate. Furthermore,
`a decrease in ureagenesis caused by partial hepatectomy did not influence acid–base balance.5 These data suggest that hepatic urea synthesis plays little
`or no role in maintaining acid–base balance, as has been proposed.6
`
`Fig. 85-1
`
`Course of blood pH, PCO2, plasma bicarbonate, and ammonium concentration of untreated OTC-deficient neonate. Early onset and persistence of
`hyperventilation and respiratory alkalosis are apparent.
`
`about:blank
`
`Page 2 of 89
`
`Page 2 of 89
`
`

`

`10/8/15, 4:28 PM
`
`A defect in the ureagenic pathway has two consequences: arginine becomes an essential amino acid7 (except in arginase deficiency, where the enzyme
`defect results in a failure of degradation of arginine) and nitrogen atoms accumulate in a variety of molecules, the pattern of which varies according to the
`specific enzymatic defect, although plasma levels of ammonium and glutamine are increased in all urea cycle disorders not under metabolic control.
`Waste Nitrogen Disposal
`The biochemical pathway of urea synthesis is described in Fig. 85-2 and in Table 85-1. Waste nitrogen disposal is far more complex, requiring interorgan,
`intrahepatic, and cellular compartmentation relationships in the conversion to urea of nitrogen not used for net biosynthetic purposes. Although it has
`been known for decades that ammonium and aspartate are the sources of nitrogen for ureagenesis, the pathways from amino acid nitrogen to ammonium
`and aspartate have been less clear.
`
`Fig. 85-2
`
`Substrates, products, and cofactors required for ureagenesis. The asterisks denote waste nitrogen atoms. AS = argininosuccinic acid synthetase; AL =
`argininosuccinase; CPS = carbamyl phosphate synthetase; NAGS = N-acetylglutamate synthetase; OTC = ornithine transcarbamylase.
`
`Table 85-1
`
`Table 85-1: The Enzymes of the Urea Cycle*
`
`about:blank
`
`Page 3 of 89
`
`Page 3 of 89
`
`

`

`10/8/15, 4:28 PM
`
`Intrahepatic Sources of Nitrogen for Ureagenesis
`
`It was proposed on theoretical grounds15 that intramitochondrial ammonium for the carbamyl phosphate synthetase (CPS) reaction was derived from the
`oxidative deamination of glutamate by glutamate dehydrogenase (Fig. 85-3). Although this interpretation is commonly accepted, attempts to verify this
`hypothesis in respiring mitochondria have repeatedly shown that the vast portion of glutamate is not deaminated but rather transaminated,16, 17
`suggesting that glutamate may not be the principal precursor for citrulline biosynthesis. The virtual absence of experimental evidence supporting the
`hypothesis that oxidative deamination of glutamate via glutamate dehydrogenase is a source of ammonium for the biosynthesis of citrulline and urea has
`led some researchers to conclude that, “studies of glutamate dehydrogenase in liver have failed to yield any clear consensus of the role of this enzyme”18
`or “it is still not possible to define the role of this enzyme in animal tissues.”19 Krebs also has reviewed this subject.20
`
`Fig. 85-3
`
`Pathways of waste nitrogen synthesis from amino acids. Muscle, by virtue of its production of alanine and glutamine, is the major source of nitrogen
`destined for incorporation into urea. The role of the intestines, kidney, and liver are outlined as described in the text.
`
`about:blank
`
`Page 4 of 89
`
`Page 4 of 89
`
`

`

`10/8/15, 4:28 PM
`
`Jungermann21 reviewed the role of metabolic zonation in the liver as it pertains to nitrogen homeostasis. It is proposed that periportal hepatocytes
`predominantly contain enzymes that catalyze transamination reactions and ureagenesis, whereas perivenous hepatocytes predominantly contain enzymes
`that catalyze the amidation or deamination of glutamate to glutamine or ammonium and ketoglutarate, respectively.
`Cooper et al.22 suggested that no more than 20 percent (and possible much less) of the α-amino moiety of liver glutamate is deaminated in vivo, but
`rather it is predominantly transaminated to aspartate and incorporated into urea. These studies were done in a series of three experiments in which each of
`13N-labeled glutamate, alanine, and glutamine (amide) was injected into the portal vein, after which the liver was freeze-clamped at intervals of 5 to 60 s
`and the distribution of the label described. Aspartate and urea were promptly labeled after 13N-alanine and 13N-glutamate were injected but not after 13N-
`glutamine (amide). Ammonium and citrulline were not labeled, suggesting that little glutamate was deaminated. The absence of incorporation of
`glutamine nitrogen into the urea cycle in these in vivo experiments does not support the glutamine channeling hypothesis of Meijer.23 The rapidity of
`nitrogen exchange among the linked transaminases in these and other studies22 was striking—within 10 s of the injection of the labeled amino acids or
`ammonium. In previous studies24 these investigators showed that intraportal vein injection of 13N-ammonium resulted in labeling of citrulline.
`From these studies it may be concluded that although glutamate may be deaminated, it may not be a major source of ammonium for the CPS reaction. As
`described below, extrahepatic glutamine metabolism provides the single most important source of ammonium for the CPS reaction. However, within the
`liver there are a number of other amino acids that are deaminated and may provide ammonium for ureagenesis (e.g., histidine, tryptophan, threonine, and
`lysine).
`Extrahepatic Sources of Nitrogen for Ureagenesis
`
`Intestine.
`
`In a series of studies of the metabolism of the perfused rat intestine, Windmueller and Spaeth25, 26 showed not only that glutamine carbon atoms were an
`important respiratory fuel but also that glutamine nitrogen was converted to the urea precursors ammonium, citrulline, and alanine, all of which were
`released into the portal circulation (Fig. 85-3). Whereas alanine and ammonium are taken up by the liver, citrulline apparently is not but rather is
`transported to the kidney, where it is converted to arginine.27
`
`Kidney.
`
`Rat kidney uptake of citrulline is approximately equivalent to its rate of intestinal release.27, 28 This observation is compatible with the report that
`
`about:blank
`
`Page 5 of 89
`
`Page 5 of 89
`
`

`

`10/8/15, 4:28 PM
`
`anephric animals incorporate little citrulline into liver.29 In confirmation of the absence of a large role of the liver in citrulline metabolism are the findings
`in patients with urea cycle disorders who have undergone orthotopic liver transplantation; plasma citrulline levels are undetectable or nearly so, similar to
`the pretransplant values.30, 31
`That the kidney may be an extrahepatic source of ammonium is suggested by the observation that the ammonium concentration of the renal vein exceeds
`that of the renal artery; a consequence of renal glutaminase activity. Thus, the kidney may supply ammonium directly for the CPS reaction and, as
`described above, generate another waste nitrogen atom by catalyzing the synthesis of arginine from citrulline and aspartate via renal argininosuccinic acid
`synthetase (AS).
`
`Muscle.
`Because glutamine is constantly being extracted from the circulation by the intestine, a potent source of glutamine must be found elsewhere. The most
`likely source of glutamine is muscle; several studies have shown a net release of glutamine from muscle in vivo 32, 33 and in vitro.34 The biosynthetic
`pathway for muscle glutamine synthesis is not entirely clear, although glutamine synthetase does play a role.32 The source of ammonium for amidation is
`also unclear; purine nucleotide deamination is a possibility.35, 36 Alanine production by muscle, via transamination of pyruvate, represents another
`important nitrogen precursor for ureagenesis.
`Figure 85-3 presents an integrated view of the interorgan relationships required for synthesis of urea. Muscle appears to be the starting point of waste
`nitrogen disposal via transamination of amino acid nitrogen to alanine and glutamate and thence amidation of glutamate to produce glutamine.
`The Biochemistry of the Urea Cycle
`The enzymes, substrates, and cofactors required for ureagenesis are described in Fig. 85-2 and Tables 85-1 and 85-2. CPS, a mitochondrial matrix
`enzyme, catalyzes the biosynthesis of carbamyl phosphate (CP) from ammonium and bicarbonate; N-acetylglutamate [NAG; synthesized from glutamate
`and acetyl coenzyme A (CoA)] is an allosteric cofactor for this enzyme and may be an important regulator of ureagenesis. OTC, also a mitochondrial
`matrix enzyme, catalyzes the biosynthesis of citrulline from ornithine and CP (see Chapter 83 for a discussion of the mitochondrial import of ornithine).
`Citrulline is exported to the cytosol, where it condenses with aspartate via AS to form argininosuccinate, which is cleaved to arginine and fumarate by
`argininosuccinase. Arginine is subsequently hydrolyzed by arginase to urea and ornithine, the latter again to be transcarbamylated to citrulline.
`Table 85-2: Concentration of Urea Cycle Intermediates in Rat Liver
`μmol per g wet weight
`Carbamyl phosphate 0.001
`Ornithine
`0.2–0.6
`Citruline
`0.03–0.1
`Aspartate
`0.3–3.5
`Argininosuccinate
`0.034
`Arginine
`0.02–0.1
`
`SOURCE: Data taken from Meijer and Hensgens,10 except for carbamyl phosphate, which was taken from Cooper et al.24
`Studies of urea cycle enzyme activities in the human fetus reveal activity by 10 to 13 weeks of gestation; at approximately 20 weeks of gestation, enzyme
`activity is similar to that found at birth, which may be 50 to 90 percent of adult values.37, 38
`Although the mutant enzymes are mainly characterized by a reduction of their activity under all conditions, a number of other biochemical characteristics
`have been reported, principally for OTC. K m mutants for both substrates have been found, as well as sensitivity to other factors, pH, temperature, and
`substrates.39–46
`Also relevant to inborn errors of ureagenesis is the understanding that the α and the ω nitrogen atoms of ornithine do not normally serve as waste
`nitrogen products. Only ammonium nitrogen and aspartate nitrogen (Fig. 85-2) (derived from the free amino acid pool) constitute waste nitrogen atoms,
`which are incorporated into urea, argininosuccinate, and arginine.
`Regulation of Ureagenesis
`In humans the synthesis of urea is a function of nitrogen intake. On high nitrogen intakes (100 g protein), urinary urea nitrogen accounts for over 80
`percent of dietary nitrogen, whereas on low nitrogen intakes (42 g protein), urinary urea nitrogen accounts for 46 percent of dietary nitrogen. The
`physiologic, biochemical, and molecular mechanisms accounting for this regulation are incompletely understood.47
`Although it has been demonstrated in rats48 and primates49 that there is a coordinated increase in all hepatic urea cycle enzyme activities when dietary
`nitrogen is increased, the regulatory factors accounting for these specfic events are complex. The quality of dietary nitrogen and energy intake may play a
`role,50 perhaps by affecting transcriptional factors.51, 52 Hormonal factors also play a role.47, 52, 54 Much attention has been focused on the role of small
`
`about:blank
`
`Page 6 of 89
`
`Page 6 of 89
`
`

`

`10/8/15, 4:28 PM
`
`molecular weight substrates (ammonia, pH, and amino acids, especially arginine, ornithine, and glutamine). NAG, the product of glutamate and acetyl
`CoA via NAG synthetase, an intramitochondrial matrix enzyme (Fig. 85-2), is known to be an arginine-sensitive allosteric activator of CPS.9, 55–58
`Molecular Analysis of Urea Cycle Enzymes Genbank Accession
`Number:
`Cps1:
`Human cDNA NM_001875
`OTC:
`Human cDNAs K02100, D00230
`Human exons D00221, D00229
`Mouse cDNA, includes Spf/ash M17030
`AS:
`Human cDNA NM_000050
`Human exons L00079, L00084
`Human Exons AH002610
`AL:
`Human cDNAs NH_000048, M57638
`Arginase 1:
`Human cDNA NM_000045
`Human exons X12662, X12669
`Arginase 2:
`Human cDNAs NM_001172, U82256, D86724, U75667
`Carbamyl Phosphate Synthetase
`Molecular analysis of mammalian CPS I has enabled prediction of the structure of this large hepatic mitochondrial enzyme and revealed it to be the
`product of fusion of two ancestral functional domains that sequentially catalyze glutamine amide transfer and synthesis of CP. The contemporary
`mammalian enzyme functions as a dimer of 160-kDa subunits. In liver this protein constitutes as much as 15 to 30 percent of mitochondrial protein and 4
`percent of total cell protein. This is a feature of stability of the enzyme, rather than a high rate of synthesis; the messenger RNA (mRNA) is of moderately
`low abundance. However, because the mRNA encoding the subunit is necessarily large, enrichment for it was accomplished in a straightforward manner
`using sucrose gradient fractionation of polyA+ mRNA from rat liver.59 A complementary DNA (cDNA) clone synthesized from the enriched mRNA was
`identified by hybrid-selected translation, and this clone was in turn used to identify additional rat and human cDNA clones. Northern analysis using these
`cDNAs identifies a 5-kb mRNA in liver. Developmental analysis of CPS mRNA in rat liver revealed its first appearance late in gestation at day 17, an
`increase to 30 to 40 percent of adult levels over the following 3 to 4 days, then a decline at the time of birth followed by a slow increase over a 3-week
`period to adult levels.
`Treatment of rats with dibutyryl cyclic adenosine monophosphate (cAMP) or dexamethasone led to a twofold increase of CPS RNA in liver, and
`starvation for 5 days led to a 37-fold increase in RNA.60 When levels of CPS RNA were examined in rat hepatoma cells, the line 5123D was found to
`contain levels twofold higher than in normal adult liver, whereas the line 3924A was devoid of CPS RNA.61
`Sequence analysis of the cloned rat CPS cDNA indicates that the corresponding mRNA contains a 5!-untranslated sequence of 139 bases, an open reading
`frame of 4500 nucleotides, and a 3!-untranslated sequence of 905 nucleotides, followed by a polyA tract.62 The rat cDNA was used as a hybridization
`probe to isolate human cDNA clones that exhibited similar-sized untranslated regions and encoded a subunit precursor with 94 percent identity to the rat
`subunit.63 The coding sequences for the CPS I subunit precursors encode at the N-terminus 38 residues that comprise a cleavable leader peptide that
`directs the precursor to mitochondria. When this coding domain and 55 codons from the mature portion of the rat cDNA were joined with the distal two
`thirds of the coding sequence for the mature subunit of OTC, the in vitro–synthesized hybrid protein was directed to isolated mitochondria64 (see later
`section on Mitochondrial Import). Like other mitochondrial signal peptides analyzed to date, the CPS leader is highly basic in overall amino acid
`
`about:blank
`
`Page 7 of 89
`
`Page 7 of 89
`
`

`

`10/8/15, 4:28 PM
`
`composition, containing four arginine residues, four lysine residues, and a single aspartate residue.
`
`Mature Subunit: An Evolutionary Fusion.
`The mature portion of the rat CPS I precursor is strikingly homologous along its length to the CPS enzyme from Escherichia coli and yeast (both termed
`type II), with 42 percent and 45 percent identity at the amino acid level, respectively,65, 66 and to the enzyme from shark liver (type III), with 72 percent
`identity.67 The type II enzymes are composed of two different subunits encoded by separate genes (Fig. 85-4). The small subunit catalyzes transfer of the
`amide nitrogen from glutamine, as ammonia, to a catalytic center for CP synthesis located on the large subunit (see Fig. 85-5 for partial reactions). The
`large subunit is composed of two homologous halves, the apparent product of an ancient gene duplication.69 By itself, this subunit can catalyze synthesis
`of CP from ammonia, bicarbonate, and adenosine triphosphate (ATP).65 The shark and mammalian enzymes (types III and I, respectively) represent a
`precise gene fusion of the glutamine amide transfer domain corresponding to the small subunit, at the N-terminus, with the synthetase domain,
`corresponding to the large subunit, at the C-terminus.62, 66 Whereas the shark enzyme, like the bacterial and fungal, employs the amide of glutamine as a
`nitrogen donor, the mammalian type I enzyme uses ammonium. Accordingly, whereas the glutamine hydrolytic site in E. coli, yeast, and shark contains a
`reactive cysteine residue,67, 70 in the mammalian enzyme, which fails to catalyze glutamine hydrolysis, the cysteine is absent. Although the hydrolytic
`site is lost, two ATP-binding sites present in the CPS catalytic centers of type II and III enzymes are retained in the mammalian enzyme and are highly
`conserved in sequence. Mutation of one of these, involving the substitution E841K, abolished synthesis of CP.71 Bicarbonate-dependent ATP hydrolysis
`was stimulated, whereas synthesis of ATP from adenosine diphosphate (ADP) and CP was suppressed (Figs. 85-4 and 85-5). E841 was thus suggested to
`be essential to phosphorylation of carbamate. The effect on HCO3 −-dependent ATPase activity suggested interaction between the two catalytic ATP sites,
`coupling formation of enzyme-bound carbamate with its phosphorylation.71 The catalytic ATP sites were tentatively localized in the rat liver enzyme
`using the ATP analogue 5!-fluoro-sulfonylbenzoyladenosine.72 Two sites were located, one in each of the duplicated synthetase domains, and were
`differentially affected by the allosteric activator NAG. Likewise, an ultraviolet photoaffinity labeling study by the same group using 32P-ATP labeled the
`two synthetase domains73 (see also Guy and Evans74 for a study of the homologous synthetase subdomains of CPS II, which, remarkably, confer CPS
`activity when joined individually with the amide transfer domain, but they form noncovalent dimers).
`
`Fig. 85-4
`
`Evolution of CPS enzymes. Steps are shown of gene duplication (open bar), fusion, signal attachment (black bar), loss of active site sulfhydryls (SH), and
`acquisition of N-acetylglutamate allosteric activation sites (AcGlu) that have produced the various modern-day enzymes. Note that, consistent with the
`immediate relationship of types III and I CPS enzymes (lower two bars), the intron–exon organization of CPS III from the pufferfish (fugu) and from rat
`CPS I have been found to be identical.68 Notably, however, the fugu gene spans only 21 kb, whereas the rat spans 88 kb. (Reprinted with permission from
`Hong J, Salo WL, Lusty CJ, Anderson PM: Carbamyl phosphate synthetase III, an evolutionary intermediate in the transition between glutamine-
`dependent and ammonia-dependent carbamyl phosphate synthetases. J Mol Biol 243:131, 1994.)
`
`Fig. 85-5
`
`about:blank
`
`Page 8 of 89
`
`Page 8 of 89
`
`

`

`Partial reactions of carbamyl phosphate synthetase enzymes. In the first reaction, performed by the amide transfer domain (see Fig. 85-4, glutaminase),
`glutamine is hydrolyzed to produce ammonia, which is then transferred to the other domain, the CPS catalytic center, for the third reaction (ammonia
`reaction). Note that the first reaction is mediated by CPS II and III enzymes, whereas CPS I uses ammonium as a donor and does not perform glutamine
`hydrolysis (although, nevertheless, CPS I has preserved its amide transfer domain). In the second reaction (activation), performed by the catalytic
`domain, bicarbonate is activated by ATP to form carboxy phosphate, a transient intermediate, which in turn reacts with ammonia in the third (ammonia)
`reaction to form carbamate, which in the fourth reaction (phosphorylation) is phosphorylated to produce carbamyl phosphate. Two ATPs are consumed:
`one in activation and one in phosphorylation of carbamate. (Adapted with permission from Guy HI, Evans DR: Function of the major synthetase
`subdomains of carbamyl-phosphate synthetase. J Biol Chem 271:13762, 1996.)
`
`10/8/15, 4:28 PM
`
`Carbamyl phosphate synthetase enzymes are subject to allosteric regulation. In the case of the E. coli enzyme, the regulators are uridine monophosphate
`(UMP), inosine monophosphate (IMP), and cytidine monophosphate (CMP), and competition for these regulators was examined using a fluorescent
`analogue.75 Photoaffinity labeling with 14C UMP identified a specific lysine.76 The mammalian mitochondrial enzyme, as well as the type III fish
`enzyme, is activated by NAG. A photoaffinity compound, N-chloroacetyl L-[14C]glutamate, could be bound to the mammalian mitochondrial enzyme
`and was competed by NAG. The site of binding could be localized after irradiation, using limited proteolysis, to the C-terminal 20 kDa.77 This argues
`that the NAG binding site is not evolved from the N-terminally situated glutamine substrate binding site (corresponding to the small subunit in E. coli)
`(however, see McCudden and Powers-Lee,78 who observed carbodiimide-activated 14C-NAG to identify multiple sites including two in the N-terminus
`and two near the ATP sites). Structural requirements on the part of NAG for binding have been examined,77, 79 and, in general, analogues binding with
`high affinity acted as activators.
`
`Structure of E.ColiCPS
`A major recent advance in mechanistic understanding of CPS comes from the determination of the crystal structure of the E.coli enzyme at 2.1 Å
`resolution by Holden, Rayment, and their colleagues.600 This reveals an α/β heterodimer with an amidotransferase subunit of 42 kDa and a larger
`synthetase subunit of 118 kDa, itself divided into four domains (Fig. 85-5a). In the small subunit, a catalytic triad composed of a cysteine,269 a glutamate,
`and a histidine mediates the hydrolysis of glutamine to produce an NH3 molecule. This product appears to transfer through a tunnel identified between
`the small subunit active site and the carboxy phosphate synthetic site in the large subunit. This latter site in the crystal structure contains resolvable ADP
`and Pi; presumably the terminal phosphate marks the site of the formation of carboxy phosphate. This site, able to stabilize carboxy phosphate, may also
`function as the site for the reaction with ammonia, forming carbamate. The carbamate product must then in turn transfer through a second limb of the
`identifiable tunnel (>100 Å in net length) to reach the carbamyl phosphate synthetic site. While these two phosphorylation sites, one producing carboxy
`
`about:blank
`
`Page 9 of 89
`
`Page 9 of 89
`
`

`

`10/8/15, 4:28 PM
`
`phosphate and the other producing carbamyl phosphate, lie in subdomains with shared primary structure and essentially the same structural fold (with the
`subdomains related to each other by a local two-fold symmetry axis), the subdomains differ at the active sites. For example, the terminal phosphate is not
`visible in the carbamyl phosphate synthetic site. The positions of critical metals in these active sites also differ. Two additional domains also comprise the
`large subunit, an oligomerization domain, enabling conversion of the αβ heterodimer to (αβ)4 tetramers, and the allosteric domain, binding IMP via a
`Rossmann fold or ornithine via a different site, but also contributing to oligomerization.
`
`Transcriptional Regulation.
`
`The human CPS I gene has been localized to chromosome 2q35 by fluorescence in situ hybridization.80, 81 Initial analysis of the 5! flanking portion of the
`gene indicated a TATA sequence at −30 bases.82, 83 A flanking promoter region between −34 and −150 has been observed to contain four functional
`segments: an element rich in GAG (−34 to −68), composed of a direct repeat of the sequence GAGGAGGGG; and three adjoining elements (−79 to
`−150), termed I, II, and III.84 In transient transfections of H4IIEC3 rat hepatoma cells, the GAG element was found to be sufficient to activate