Harper's Biochemistry - Catabolism of Proteins & Amino Acid Nitrogen Chapter 28 PDF

C H A P T E R Catabolism o Proteins & o Amino Acid Nitrogen Victor W. Rodwell, PhD 28 O B J E C TI V E S Describe protein turnover, indicate the mean rate o pro...

C H A P T E R Catabolism o Proteins & o Amino Acid Nitrogen Victor W. Rodwell, PhD 28 O B J E C TI V E S Describe protein turnover, indicate the mean rate o protein turnover in healthy individuals, and provide examples o human proteins that are degraded at rates After studying this chapter, greater than the mean rate. you should be able to: Outline the events in protein turnover by both ATP-dependent and ATP- independent pathways, and indicate the roles in protein degradation played by the proteasome, ubiquitin, cell surace receptors, circulating asialoglycoproteins, and lysosomes. Indicate how the ultimate end products o nitrogen catabolism in mammals dier rom those in birds and fsh. Illustrate the central roles o transaminases (aminotranserases), o glutamate dehydrogenase, and o glutaminase in human nitrogen metabolism. Use structural ormulas to represent the reactions that convert NH3, CO2, and the amide nitrogen o aspartate into urea, and identiy the subcellular locations o the enzymes that catalyze urea biosynthesis. Indicate the roles o allosteric regulation and o acetylglutamate in the regulation o the earliest steps in urea biosynthesis. Explain why metabolic deects in dierent enzymes o urea biosynthesis, although distinct at the molecular level, present similar clinical signs and symptoms. Describe both the classical approaches and the role o tandem mass spectrometry in screening neonates or inherited metabolic diseases. BIOMEDICAL IMPORTANCE o amino acids. issues thereore convert ammonia to the amide nitrogen o the nontoxic amino acid gutamine. Subse- In norma aduts, nitrogen intake matches nitrogen excreted. quent deamination o gutamine in the iver reeases ammonia, Positive nitrogen baance, an excess o ingested over excreted which is eicienty converted to urea, which is not toxic. How- nitrogen, accompanies growth and pregnancy. Negative nitro- ever, i iver unction is compromised, as in cirrhosis or hepa- gen baance, where output exceeds intake, may oow surgery, titis, eevated bood ammonia eves generate cinica signs and advanced cancer, and the nutritiona disorders kwashiorkor symptoms. Each enzyme o the urea cyce provides exampes and marasmus. Genetic disorders that resut rom deects in o metaboic deects and their physioogic consequences. In the genes that encode ubiquitin, ubiquitin igases, or deubiq- addition, the urea cyce provides a useu moecuar mode or uitinating enzymes that participate in the degradation o cer- the study o other human metaboic deects. tain proteins incude Angeman syndrome, juvenie Parkinson disease, von Hippe-Lindau syndrome, and congenita poy- cythemia. his chapter describes how the nitrogen o amino PROTEIN TURNOVER acids is converted to urea, and the metaboic disorders that he continuous degradation and synthesis (turnover) o ce- accompany deects in this process. Ammonia, which is highy uar proteins occur in a orms o ie. Each day, humans turn toxic, arises in humans primariy rom the α-amino nitrogen over 1 to 2% o their tota body protein, principay musce 279 280 SECTION VI Metabolism o Proteins & Amino Acids protein. High rates o protein degradation occur in tissues N-terminus that are undergoing structura rearrangement, or exampe, uterine tissue during pregnancy, skeeta musce in starvation, Lysine 63 and tadpoe tai tissue during metamorphosis. Whie approxi- matey 75% o the amino acids iberated by protein degrada- tion are reutiized, the remaining excess ree amino acids are not stored or uture use. Amino acids not immediatey incor- porated into new protein are rapidy degraded. he major por- C-terminus tion o the carbon skeetons o the amino acids is converted to Lysine 48 amphiboic intermediates, whie in humans the amino nitro- gen is converted to urea and excreted in the urine. FIGURE 28–1 Three-dimensional structure of ubiquitin. Shown are α-helices (blue), β-strands (green), and the R-groups o lysyl residues (orange). Lys48 & Lys63 are sites or attachment PROTEASES & PEPTIDASES o additional ubiquitin molecules during polyubiquitination. (Rogerdodd/Wikipedia) DEGRADE PROTEINS TO AMINO ACIDS he reative susceptibiity o a protein to degradation is expressed avor ubiquitination. Attachment o a singe ubiquitin moecue as its haf-ife (t1/2), the time required to ower its concentration to transmembrane proteins aters their subceuar ocaization to ha o its initia vaue. Ha-ives o iver proteins range rom and targets them or degradation. Soube proteins undergo under 30 minutes to over 150 hours. ypica “housekeeping” poyubiquitination, the igase-catayzed attachment o our or enzymes such as those o gycoysis, have t1/2 vaues o over more additiona ubiquitin moecues (Figure 28–1). Subsequent 100 hours. By contrast, key reguatory enzymes may have t1/2 degradation o ubiquitin-tagged proteins takes pace in the vaues as ow as 0.5 to 2 hours. PES sequences, regions rich in proteasome, a macromoecue that aso is ubiquitous in proine (P), gutamate (E), serine (S), and threonine (), target some proteins or rapid degradation. Intraceuar proteases hydroyze interna peptide bonds. he resuting peptides are then degraded to amino acids by endopeptidases that hydro- yze interna peptide bonds, and by aminopeptidases and carboxypeptidases that remove amino acids sequentiay rom the amino- and carboxy-termini, respectivey. ATP-Independent Degradation Degradation o bood gycoproteins (see Chapter 46) oows oss o a siaic acid moiety rom the nonreducing ends o their oigosaccharide chains. Asiaogycoproteins are then interna- ized by iver-ce asiaogycoprotein receptors and degraded by ysosoma proteases. Extraceuar, membrane-associated, and ong-ived intraceuar proteins are aso degraded in yso- somes by AP-independent processes. ATP & Ubiquitin-Dependent Degradation Degradation o reguatory proteins with short ha-ives and o abnorma or misoded proteins occurs in the cytoso, and requires AP and ubiquitin. Named based on its presence in a eukaryotic ces, ubiquitin is a sma (8.5 kDa, 76 residue) poypep- tide that targets many intraceuar proteins or degradation. he FIGURE 28–2 Reactions involved in the attachment of primary structure o ubiquitin is highy conserved. Ony 3 o 76 ubiquitin (Ub) to proteins. Three enzymes are involved. E1 is an acti- residues dier between yeast and human ubiquitin. Figure 28–1 vating enzyme, E2 a transerase, and E3 a ligase. While depicted as iustrates the three-dimensiona structure o ubiquitin. Ubiquitin single entities, there are several types o E1, and over 500 types o E2. moecues are attached by non–α-peptide bonds ormed between The terminal COOH o ubiquitin irst orms a thioester. The coupled the carboxy termina o ubiquitin and the ε-amino groups o ysy hydrolysis o PPi by pyrophosphatase ensures that the reaction will proceed readily. A thioester exchange reaction now transers acti- residues in the target protein (Figure 28–2). he residue present vated ubiquitin to E2. E3 then catalyzes the transer o ubiquitin to at its amino terminus aects whether a protein is ubiquitinated. the ε-amino group o a lysyl residue o the target protein. Additional Amino termina Met or Ser residues retard, whereas Asp or Arg rounds o ubiquitination result in subsequent polyubiquitination.  CHAPTER 28 Catabolism o Proteins & o Amino Acid Nitrogen 281 Ub Ub Ub Ub Regulatory particle Gated pore Core particle Active sites FIGURE 28–4 An end-on view of a proteasome. Gated (Thomas Splettstoesser/Wikipedia) Regulatory pore particle utiization by various tissues. Musce generates over ha o the tota body poo o ree amino acids, and iver is the site o the FIGURE 28–3 Representation of the structure of a protea- urea cyce enzymes necessary or disposa o excess nitrogen. some. The upper ring is gated to permit only polyubiquitinated pro- teins to enter the proteosome, where immobilized internal proteases Musce and iver thus pay major roes in maintaining circuat- degrade them to peptides. ing amino acid eves. Figure 28–5 summarizes the postabsorptive state. Free amino acids, particuary aanine and gutamine, are reeased eukaryotic ces. he proteasome consists o a macromoecuar, rom musce into the circuation. Aanine is extracted primar- cyindrica compex o proteins, whose stacked rings orm a cen- iy by the iver, and gutamine is extracted by the gut and the tra pore that harbors the active sites o proteoytic enzymes. For kidney, both o which convert a signiicant portion to aanine. degradation, a protein thus must irst enter the centra pore. Entry Gutamine aso serves as a source o ammonia or excretion into the core is reguated by the two outer rings that recognize by the kidney. he kidney provides a major source o serine poyubiquitinated proteins (Figures 28–3 and 28–4). or uptake by periphera tissues, incuding iver and musce. For the discovery o ubiquitin-mediated protein degradation, Aaron Ciechanover and Avram Hershko o Israe and Irwin Rose o the United States were awarded the 2004 Nobe Prize in Kidney Chemistry. Genetic disorders that resut rom deects in the genes NH3 that encode ubiquitin, ubiquitin igases, or deubiquitinating Brain enzymes incude Angeman syndrome, autosoma recessive juvenie Parkinson disease, von Hippe-Lindau syndrome, and congenita poycythemia. For additiona aspects o protein degradation and o ubiquitination, incuding its roe in the ce Val Ser cyce, see Chapters 4 and 35. Gut Ala Gln Ala INTERORGAN EXCHANGE Urea MAINTAINS CIRCULATING LEVELS Ala Glucose OF AMINO ACIDS Muscle Liver he maintenance o steady-state concentrations o circuat- FIGURE 28–5 Interorgan amino acid exchange in normal ing pasma amino acids between meas depends on the net postabsorptive humans. The key role o alanine in amino acid out- baance between reease rom endogenous protein stores and put rom muscle and gut and uptake by the liver is shown. 282 SECTION VI Metabolism o Proteins & Amino Acids state, they provide the brain with an energy source, and post- Liver Blood Muscle prandiay they are extracted predominanty by musce, having Glucose been spared by the iver. ANIMALS CONVERT α-AMINO Glucose Glucose NITROGEN TO VARIED END Urea PRODUCTS Pyruvate Pyruvate – NH2 Depending on their ecoogica niche and physioogy, dier- –NH2 ent animas excrete excess nitrogen as ammonia, uric acid, or Alanine Alanine urea. he aqueous environment o teeostean ish, which are Amino acids ammonoteic (excrete ammonia), permits them to excrete Alanine water continuousy to aciitate excretion o ammonia, which is highy toxic. Whie this approach is appropriate or an aquatic anima, birds must both conserve water and maintain ow weight. Birds, which are uricoteic, address both prob- ems by excreting nitrogen-rich uric acid (see Figure 33–11) as semisoid guano. Many and animas, incuding humans, FIGURE 28–6 The glucose-alanine cycle. Alanine is synthe- are ureoteic and excrete nontoxic, highy water-soube urea. sized in muscle by transamination o glucose-derived pyruvate, Since urea is nontoxic to humans, high bood eves in rena released into the bloodstream, and taken up by the liver. In the liver, the carbon skeleton o alanine is reconverted to glucose and disease are a consequence, not a cause, o impaired rena released into the bloodstream, where it is available or uptake by unction. muscle and resynthesis o alanine. BIOSYNTHESIS OF UREA Branched-chain amino acids, particuary vaine, are reeased Urea biosynthesis occurs in our stages: (1) transamination, by musce and taken up predominanty by the brain. (2) oxidative deamination o gutamate, (3) ammonia trans- Aanine is a key guconeogenic amino acid (Figure 28–6). port, and (4) reactions o the urea cyce (Figure 28–8). he he rate o hepatic guconeogenesis rom aanine is ar higher expression in iver o the RNAs or a the enzymes o the urea than rom a other amino acids. he capacity o the iver or cyce increases severaod in starvation, probaby secondary guconeogenesis rom aanine does not reach saturation unti to enhanced protein degradation to provide energy. the aanine concentration reaches 20 to 30 times its norma physioogic eve. Foowing a protein-rich mea, the spanchnic tissues reease amino acids (Figure 28–7) whie the periphera Transamination Transfers α-Amino musces extract amino acids, in both instances predominanty Nitrogen to α-Ketoglutarate, branched-chain amino acids. Branched-chain amino acids Forming Glutamate thus serve a specia roe in nitrogen metaboism. In the asting ransamination reactions interconvert pairs o α-amino acids and α-keto acids (Figure 28–9). ransamination reac- Kidney tions, which are reey reversibe, aso unction in amino acid Brain Ala (20 % Val amBran Po ino ch Gln Gut r ta ac ed- lc ids ch irc ) ain ula tio n (60% Branched-chain amino acids) Muscle Liver Ala FIGURE 28–7 Summary of amino acid exchange between FIGURE 28–8 Overall flow of nitrogen in amino acid organs immediately after feeding. catabolism.  CHAPTER 28 Catabolism o Proteins & o Amino Acid Nitrogen 283 NH +3 O R COO– CH CH O– C O– R1 C R1 C N O O HO CH2 OPO3–2 O NH + 3 H3C N C O– CH O– R2 C R2 C O O FIGURE 28–11 Structure of a Schiff base formed between pyridoxal phosphate and an amino acid. FIGURE 28–9 Transamination. The reaction is reely reversible with an equilibrium constant close to unity. associated with eevated serum eves o aminotranserases (see abe 7–1). biosynthesis (see Figure 27–4). A o the common amino acids except ysine, threonine, proine, and hydroxyproine partici- pate in transamination. ransamination is not restricted to l-GLUTAMATE DEHYDROGENASE α-amino groups. he δ-amino group o ornithine (but not the OCCUPIES A CENTRAL POSITION ε-amino group o ysine) readiy undergoes transamination. Aanine-pyruvate aminotranserase (aanine aminotrans- IN NITROGEN METABOLISM erase, EC 2.6.1.2) and gutamate-α-ketogutarate aminotrans- ranser o amino nitrogen to α-ketogutarate orms l-gutamate. erase (gutamate aminotranserase, EC 2.6.1.1) catayze the Hepatic l-gutamate dehydrogenase (GDH), which can use transer o amino groups to pyruvate (orming aanine) or to either NAD+ or NADP+, reeases this nitrogen as ammonia α-ketogutarate (orming gutamate). (Figure 28–12). Conversion o α-amino nitrogen to ammo- Each aminotranserase is speciic or one pair o substrates, nia by the concerted action o gutamate aminotranserase and but nonspeciic or the other pair. Since aanine is aso a sub- GDH is oten termed “transdeamination.” Liver GDH activ- strate or gutamate aminotranserase, the α-amino nitrogen ity is aostericay inhibited by AP, GP, and NADH, and is rom a amino acids that undergo transamination can be con- activated by ADP. he GDH reaction is reey reversibe, and centrated in gutamate. his is important because l-gutamate aso unctions in amino acid biosynthesis (see Figure 27–1). is the ony amino acid that undergoes oxidative deamination at an appreciabe rate in mammaian tissues. he ormation o ammonia rom α-amino groups thus occurs mainy via the AMINO ACID OXIDASES REMOVE α-amino nitrogen o l-gutamate. NITROGEN AS AMMONIA ransamination occurs via a “ping-pong” mechanism l-Amino acid oxidase o iver and kidney convert an amino characterized by the aternate addition o a substrate and acid to an α-imino acid that decomposes to an α-keto acid with reease o a product (Figure 28–10). Foowing remova o its reease o ammonium ion (Figure 28–13). he reduced avin α-amino nitrogen by transamination, the remaining carbon is reoxidized by moecuar oxygen, orming hydrogen perox- “skeeton” o an amino acid is degraded by pathways discussed ide (H2O2), which then is spit to O2 and H2O by cataase, EC in Chapter 29. 1.11.1.6. Pyridoxa phosphate (PLP), a derivative o vitamin B6, is present at the cataytic site o a aminotranserases, and pays a key roe in cataysis. During transamination, PLP serves as Ammonia Intoxication Is a “carrier” o amino groups. An enzyme-bound Schi base Life-Threatening (Figure 28–11) is ormed between the oxo group o enzyme- he ammonia produced by enteric bacteria and absorbed into bound PLP and the α-amino group o an α-amino acid. he porta venous bood and the ammonia produced by tissues are Schi base can rearrange in various ways. In transamination, rapidy removed rom circuation by the iver and converted rearrangement orms an α-keto acid and enzyme-bound pyri- to urea. hus, normay, ony traces (10-20 μg/dL) are present doxamine phosphate. As noted earier, certain diseases are in periphera bood. his is essentia, since ammonia is toxic Pyr Glu Ala CHO CH2NH2 KG CH2NH2 CHO E CHO E E E CH2NH2 E E E CHO Ala Pyr KG Glu FIGURE 28–10 “Ping-pong” mechanism for transamination. E—CHO and E—CH2NH2 represent enzyme-bound pyridoxal phosphate and pyridoxamine phosphate, respectively. (Ala, alanine; Glu, glutamate; KG, α-ketoglutarate; Pyr, pyruvate.) 284 SECTION VI Metabolism o Proteins & Amino Acids NH + 3 –O CH 2 CH O– C CH 2 C O O L-Glutamate Mg-ATP NH + 4 FIGURE 28–12 The reaction catalyzed by glutamate dehy- drogenase, EC 1.4.1.2. NAD(P)+ means that either NAD+ or NADP+ Glutamine synthetase can serve as the oxidoreductant. The reaction is reversible, but strongly avors glutamate ormation. Mg-ADP H 2O + Pi NH + N 3 H2 N CH 2 CH O– C CH 2 C to the centra nervous system. Shoud porta bood bypass the iver, systemic bood ammonia may reach toxic eves. his O O occurs in severey impaired hepatic unction or the deveop- L-Glutamine ment o coatera inks between the porta and systemic veins in cirrhosis. Symptoms o ammonia intoxication incude FIGURE 28–14 Formation of glutamine, catalyzed by gluta- mine synthetase, EC 6.3.1.2. tremor, surred speech, burred vision, coma, and utimatey death. Ammonia may be toxic to the brain in part because it reacts with α-ketogutarate to orm gutamate. he resuting deiciency in neonate gutamine synthetase resuts in severe depetion o α-ketogutarate then impairs unction o the tri- brain damage, mutiorgan aiure, and death. carboxyic acid (CA) cyce in neurons. Glutaminase & Asparaginase Glutamine Synthetase Fixes Ammonia Deamidate Glutamine & Asparagine as Glutamine here are two human isoorms o mitochondria gutamin- ase, termed iver-type and rena-type gutaminase. Products Formation o gutamine is catayzed by mitochondria gutamine o dierent genes, the gutaminases dier with respect to their synthetase (Figure 28–14). Since amide bond synthesis is structure, kinetics, and reguation. Hepatic gutaminase eves couped to the hydroysis o AP to ADP and Pi, the reac- rise in response to high protein intake whie rena kidney-type tion strongy avors gutamine synthesis. During cataysis, gutaminase increases in metaboic acidosis. Hydroytic reease gutamate attacks the γ-phosphory group o AP, orming o the amide nitrogen o gutamine as ammonia, catayzed by γ-gutamy phosphate and ADP. Foowing deprotonation o gutaminase (Figure 28–15), strongy avors gutamate orma- NH4+, NH3 attacks γ-gutamy phosphate, and gutamine and tion. An anaogous reaction is catayzed by l-asparaginase Pi are reeased. In addition to providing gutamine to serve (EC 3.5.1.1). he concerted action o gutamine synthetase as a carrier o nitrogen, carbon and energy between organs and gutaminase thus catayzes the interconversion o ree (Figure 28–5), gutamine synthetase pays a major roe both in ammonium ion and gutamine. ammonia detoxiication and in acid–base homeostasis. A rare NH + 3 H2 N CH 2 CH O– C CH 2 C O O L-Glutamine H2O Glutaminase NH + 4 NH + 3 –O CH 2 CH O– C CH 2 C O O L-Glutamate FIGURE 28–15 The reaction catalyzed by glutaminase, EC FIGURE 28–13 Oxidative deamination catalyzed by l-amino 3.5.1.2. The reaction proceeds essentially irreversibly in the direction acid oxidase (l-α-amino acid:O2 oxidoreductase, EC 1.4.3.2). The o glutamate and NH4+ ormation. Note that the amide nitrogen, not α-imino acid, shown in brackets, is not a stable intermediate. the α-amino nitrogen, is removed.  CHAPTER 28 Catabolism o Proteins & o Amino Acid Nitrogen 285 Formation & Secretion of Ammonia others serve as carriers o the atoms that utimatey become urea. he major metaboic roe o ornithine, citruine, and arginino- Maintains Acid–Base Balance succinate in mammas is urea synthesis. Urea synthesis is a cycic Excretion into urine o ammonia produced by rena tubuar process. Whie ammonium ion, CO2, AP, and aspartate are con- ces aciitates cation conservation and reguation o acid– sumed, the ornithine consumed in reaction 2 is regenerated in base baance. Ammonia production rom intraceuar rena reaction 5. hus, there is no net oss or gain o ornithine, citru- amino acids, especiay gutamine, increases in metaboic aci- ine, argininosuccinate, or arginine. As indicated in Figure 28–16, dosis and decreases in metaboic akaosis. some reactions o urea synthesis occur in the matrix o the mito- chondrion, and other reactions in the cytoso. Urea Is the Major End Product of Nitrogen Catabolism in Humans Carbamoyl Phosphate Synthetase I Synthesis o 1 mo o urea requires 3 mo o AP, 1 mo each Initiates Urea Biosynthesis o ammonium ion and o aspartate, and empoys ive enzymes Condensation o CO2, ammonia, and AP to orm carbamoy (Figure 28–16). O the six participating amino acids, phosphate is catayzed by mitochondria carbamoy phos- N-acetygutamate unctions soey as an enzyme activator. he phate synthetase I (EC 6.3.4.16). A cytosoic orm o this FIGURE 28–16 Reactions and intermediates of urea biosynthesis. The nitrogen-containing groups that contribute to the ormation o urea are shaded. Reactions 1 and 2 occur in the matrix o liver mitochondria and reactions 3 , 4 , and 5 in liver cytosol. CO2 (as bicarbon- ate), ammonium ion, ornithine, and citrulline enter the mitochondrial matrix via speciic carriers (see red dots) present in the inner membrane o liver mitochondria. 286 SECTION VI Metabolism o Proteins & Amino Acids enzyme, carbamoy phosphate synthetase II, uses gutamine aminotranserase then reorms aspartate. he carbon skeeton rather than ammonia as the nitrogen donor and unctions o aspartate-umarate thus acts as a carrier o the nitrogen o in pyrimidine biosynthesis (see Figure 33–9). he concerted gutamate into a precursor o urea. action o gutamate dehydrogenase and carbamoy phosphate synthetase I thus shuttes amino nitrogen into carbamoy phosphate, a compound with high group transer potentia. Cleavage of Arginine Releases Urea & Carbamoy phosphate synthetase I, the rate-imiting Reforms Ornithine enzyme o the urea cyce, is active ony in the presence o Hydroytic ceavage o the guanidino group o arginine, cata- N-acetygutamate, an aosteric activator that enhances the yzed by iver arginase (EC 3.5.3.1), reeases urea (reaction ainity o the synthetase or AP. Synthesis o 1 mo o car- 5, Figure 28–16). he other product, ornithine, reenters iver bamoy phosphate requires 2 mo o AP. One AP serves as mitochondria and participates in additiona rounds o urea the phosphory donor or ormation o the mixed acid anhy- synthesis. Ornithine and ysine are potent inhibitors o argi- dride bond o carbamoy phosphate. he second AP provides nase, and compete with arginine. Arginine aso serves as the the driving orce or synthesis o the amide bond o carbamoy precursor o the potent musce reaxant nitric oxide (NO) in a phosphate. he other products are 2 mo o ADP and 1 mo o Ca2+-dependent reaction catayzed by NO synthetase. Pi (reaction 1, Figure 28–16). he reaction proceeds stepwise. Reaction o bicarbonate with AP orms carbony phosphate and ADP. Ammonia then dispaces ADP, orming carbamate Carbamoyl Phosphate Synthetase I and orthophosphate. Phosphoryation o carbamate by the Is the Pacemaker Enzyme of the second AP then orms carbamoy phosphate. Urea Cycle he activity o carbamoy phosphate synthetase I is determined Carbamoyl Phosphate Plus Ornithine by N-acetygutamate, whose steady-state eve is dictated by Forms Citrulline the baance between its rate o synthesis rom acety-CoA and l-Ornithine transcarbamoyase (EC 2.1.3.3) catayzes trans- gutamate and its rate o hydroysis to acetate and gutamate, er o the carbamoy group o carbamoy phosphate to orni- reactions catayzed by N-acetygutamate synthetase (NAGS) thine, orming citruine and orthophosphate (reaction 2, and N-acetygutamate deacyase (hydroase), respectivey. Figure 28–16). Whie the reaction occurs in the mitochon- Acety-CoA + l-gutamate → N-acety-l-gutamate + CoASH dria matrix, both the ormation o ornithine and the subse- N-acety-l-gutamate + H2O → l-gutamate + acetate quent metaboism o citruine take pace in the cytoso. Entry o ornithine into mitochondria and exodus o citruine rom Major changes in diet can increase the concentrations o mitochondria invoves the mitochondria inner membrane individua urea cyce enzymes 10- to 20-od. For exampe, carriers ORC1, ORC2, and SLCA25A29 (Figure 28–16). starvation eevates enzyme eves, presumaby to cope with the increased production o ammonia that accompanies enhanced Citrulline Plus Aspartate Forms starvation-induced degradation o protein. Argininosuccinate Argininosuccinate synthetase (EC 6.3.4.5) inks aspartate GENERAL FEATURES OF and citruine via the amino group o aspartate (reaction 3, Figure 28–16), which provides the second nitrogen o urea. METABOLIC DISORDERS he reaction requires AP and invoves intermediate orma- he comparativey rare, but we-characterized and medicay tion o citruy-AMP. Subsequent dispacement o AMP by devastating metaboic disorders associated with the enzymes aspartate then orms argininosuccinate. o urea biosynthesis iustrate the oowing genera principes o inherited metaboic diseases: Cleavage of Argininosuccinate Forms 1. Simiar or identica cinica signs and symptoms can accom- Arginine & Fumarate pany various genetic mutations in a gene that encodes a given enzyme or in enzymes that catayze successive reac- Ceavage o argininosuccinate is catayzed by argininosuccinate tions in a metaboic pathway. yase (EC 4.3.2.1). he reaction proceeds with retention o a three nitrogens in arginine and reease o the aspartate 2. Rationa therapy is based on an understanding o the re- skeeton as umarate (reaction 4, Figure 28–16). Subsequent evant biochemica enzyme-catayzed reactions in both nor- addition o water to umarate orms l-maate, whose subse- ma and impaired individuas. quent NAD+-dependent oxidation orms oxaoacetate. hese 3. he identiication o intermediates and o anciary prod- two reactions are anaogous to reactions o the citric acid ucts that accumuate prior to a metaboic bock provides cyce, but are catayzed by cytosolic fumarase and maate the basis or metaboic screening tests that can impicate dehydrogenase. ransamination o oxaoacetate by gutamate the reaction that is impaired.  CHAPTER 28 Catabolism o Proteins & o Amino Acid Nitrogen 287 4. Deinitive diagnosis invoves quantitative assay o the activ- suicient protein, arginine, and energy to promote growth ity o the enzyme suspected to be deective. and deveopment whie simutaneousy minimizing the meta- 5. he DNA sequence o the gene that encodes a given mutant boic perturbations. enzyme is compared to that o the wid-type gene to iden- tiy the speciic mutation(s) that cause the disease. Carbamoyl Phosphate Synthetase I 6. he exponentia increase in DNA sequencing o human N-Acetygutamate is essentia or the activity o carbamoy genes has identiied dozens o mutations o an aected phosphate synthetase I, EC 6.3.4.16 (reaction 1, Figure 28–16). gene that are benign or are associated with symptoms o Deects in carbamoy phosphate synthetase I are responsibe varying severity o a given metaboic disorder. or the reativey rare (estimated requency 1:62,000) meta- boic disease termed “hyperammonemia type 1.” METABOLIC DISORDERS ARE N-Acetylglutamate Synthetase ASSOCIATED WITH EACH N-Acetygutamate synthetase, EC 2.3.1.1 (NAGS), catayzes REACTION OF THE UREA CYCLE the ormation rom acety-CoA and gutamate o the N- Five we-documented diseases represent deects in the bio- acetygutamate essentia or carbamoy phosphate synthetase synthesis o enzymes o the urea cyce. Moecuar genetic I activity. anaysis has pinpointed the oci o mutations associated with l-Gutamate + acety-CoA → N-acety-l-gutamate + CoASH each deiciency, each o which exhibits considerabe genetic and phenotypic variabiity (Tabe 28–1). Whie the cinica and biochemica eatures o NAGS dei- Urea cyce disorders are characterized by hyperammo- ciency are indistinguishabe rom those arising rom a deect nemia, encephaopathy, and respiratory akaosis. Four o the in carbamoy phosphate synthetase I, a deiciency in NAGS ive metaboic diseases, deiciencies o carbamoy phosphate may respond to administered N-acetygutamate. synthetase I, ornithine carbamoy transerase, argininosuc- cinate synthetase, and argininosuccinate yase, resut in the Ornithine Permease accumuation o precursors o urea, principay ammonia and he hyperornithinemia, hyperammonemia, and homocitru- gutamine. Ammonia intoxication is most severe when the inuria (HHH) syndrome resuts rom mutation o the ORC1 metaboic bock occurs at reactions 1 or 2 (Figure 28–16), or gene that encodes the mitochondria membrane ornithine i citruine can be synthesized, some ammonia has aready carrier. he inabiity to import cytosoic ornithine into the been removed by being covaenty inked to an organic mitochondria matrix renders the urea cyce inoperabe, with metaboite. consequent hyperammonemia, and hyperornithinemia due Cinica symptoms common to a urea cyce disorders to the accompanying accumuation o cytosoic ornithine. In incude vomiting, avoidance o high-protein oods, intermit- the absence o its norma acceptor (ornithine), mitochondria tent ataxia, irritabiity, ethargy, and severe menta retardation. carbamoy phosphate carbamoyates ysine to homocitruine, he most dramatic cinica presentation occurs in u-term resuting in homocitruinuria. inants who initiay appear norma, then exhibit progressive ethargy, hypothermia, and apnea due to high pasma ammo- nia eves. he cinica eatures and treatment o a ive disor- Ornithine Transcarbamoylase ders are simiar. Signiicant improvement and minimization he X-chromosome–inked deiciency termed “hyperammo- o brain damage can accompany a ow-protein diet ingested nemia type 2” reects a deect in ornithine transcarbamoyase as requent sma meas to avoid sudden increases in bood (reaction 2, Figure 28–16). he mothers aso exhibit hyper- ammonia eves. he goa o dietary therapy is to provide ammonemia and an aversion to high-protein oods. Leves TABLE 28–1 Enzymes of Inherited Metabolic Disorders of the Urea Cycle Enzyme Enzyme Catalog Number OMIMa Reference Figure and Reaction Carbamoyl-phosphate synthetase 1 6.3.4.16 237300 28-13➀ Ornithine carbamoyl transerase 2.1.3.3 311250 28-13➁ Argininosuccinate synthetase 6.3.4.5 215700 28-13➂ Argininosuccinate lyase 4.3.2.1 608310 28-13➃ Arginase 3.5.3.1 608313 28-13➄ a Online Mendelian inheritance in man database: ncbi.nlm.nih.gov/omim/ 288 SECTION VI Metabolism o Proteins & Amino Acids o gutamine are eevated in bood, cerebrospina uid, and Can Metabolic Disorders Be Rectified urine, probaby as a resut o enhanced gutamine synthesis in response to eevated eves o tissue ammonia. by Gene or Protein Modification Despite resuts in anima modes using an adenovira vector to treat citruinemia, at present gene therapy provides no eec- Argininosuccinate Synthetase tive soution or human subjects. However, direct CRISPR/ In addition to patients who ack detectabe argininosuccinate Cas9-based modiication o a deective enzyme can restore synthetase activity (reaction 3, Figure 28–16), 25-od eeva- unctiona enzyme activity o cutured human puripotent tions in Km or citruine have been reported. In the resuting stem ces. citruinemia, pasma and cerebrospina uid citruine eves are eevated, and 1 to 2 g o citruine are excreted daiy. SUMMARY Argininosuccinate Lyase Human subjects degrade 1 to 2% o their body protein daiy at rates that vary widey between proteins and with physioogic Argininosuccinic aciduria, accompanied by eevated eves state. Key reguatory enzymes oen have short ha-ives. o argininosuccinate in bood, cerebrospina uid, and urine, Proteins are degraded by both AP-dependent and AP- is associated with riabe, tuted hair (trichorrhexis nodosa). independent pathways. Ubiquitin targets many intraceuar Both eary- and ate-onset types are known. he metaboic proteins or degradation. Liver ce surace receptors bind deect is in argininosuccinate yase (reaction 4, Figure 28–16). and internaize circuating asiaogycoproteins destined or Diagnosis by the measurement o erythrocyte argininosucci- ysosoma degradation. nate yase activity can be perormed on umbiica cord bood Poyubiquitinated proteins are degraded by proteases or amniotic uid ces. on the inner surace o a cyindrica macromoecue, the proteasome. Entry into the proteasome is gated by a Arginase donut-shaped protein pore that rejects entry to a but poyubiquitinated proteins. Hyperargininemia is an autosoma recessive deect in the gene Fishes excrete highy toxic NH3 directy. Birds convert NH3 to or arginase (reaction 5, Figure 28–16). Unike other urea cyce uric acid. Higher vertebrates convert NH3 to urea. disorders, the irst symptoms o hyperargininemia typicay do ransamination channes amino acid nitrogen into not appear unti age 2 to 4 years. Bood and cerebrospina uid gutamate. GDH occupies a centra position in nitrogen eves o arginine are eevated. he urinary amino acid pattern, metaboism. which resembes that o ysine-cystinuria (see Chapter 29), Gutamine synthetase converts NH3 to nontoxic gutamine. may reect competition by arginine with ysine and cysteine Gutaminase reeases NH3 or use in urea synthesis. or reabsorption in the rena tubue. NH3, CO2, and the amide nitrogen o aspartate provide the atoms o urea. Analysis of Neonate Blood by Tandem Hepatic urea synthesis takes pace in part in the mitochondria Mass Spectrometry Can Detect matrix and in part in the cytoso. Metabolic Diseases Changes in enzyme eves and aosteric reguation o Metaboic diseases caused by the absence or unctiona impair- carbamoy phosphate synthetase I by N-acetygutamate reguate urea biosynthesis. ment o metaboic enzymes can be devastating. Eary dietary intervention, however, can in many instances ameiorate the Metaboic diseases are associated with deects in each enzyme otherwise inevitabe dire eects. he eary detection o such o the urea cyce, o the ORC1 ornithine carrier, and o NAGS. metaboic diseases is thus is o primary importance. Since Te metaboic disorders o urea biosynthesis iustrate six the initiation in the United States o newborn screening pro- genera principes o a metaboic disorders. grams in the 1960s, a states now conduct metaboic screen- andem mass spectrometry is the technique o choice or ing o newborn inants. he poweru and sensitive technique screening neonates or inherited metaboic diseases. o tandem mass spectrometry (MS) (see Chapter 4) can in a ew minutes detect over 40 anaytes o signiicance in the detection o metaboic disorders. Most states empoy tandem REFERENCES MS to screen newborns to detect metaboic disorders such as Adam S, Ameida MF, Assoun M, et a: Dietary management o urea cyce disorders: European practice. Mo Genet Metab organic acidemias, aminoacidemias, disorders o atty acid 2013;110:439. oxidation, and deects in the enzymes o the urea cyce. An Burgard P, Köker S, Haege G, et a. 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