Harper's Biochemistry - Catabolism of Proteins & Amino Acid Nitrogen Chapter 28 PDF
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Victor W. Rodwell, PhD
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This chapter of Harper's Biochemistry details the catabolism of proteins and amino acid nitrogen. It explains protein turnover, the roles of enzymes in nitrogen metabolism, and the urea cycle. The chapter also discusses metabolic disorders related to these processes.
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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 surace receptors, circulating asialoglycoproteins, and lysosomes. Indicate how the ultimate end products o nitrogen catabolism in mammals dier rom those in birds and fsh. Illustrate the central roles o transaminases (aminotranserases), 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 identiy 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 deects in dierent 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 thereore convert ammonia to the amide nitrogen o the nontoxic amino acid gutamine. Subse- In norma aduts, nitrogen intake matches nitrogen excreted. quent deamination o gutamine in the iver reeases ammonia, Positive nitrogen baance, an excess o ingested over excreted which is eicienty 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 baance, where output exceeds intake, may oow surgery, titis, eevated bood ammonia eves generate cinica signs and advanced cancer, and the nutritiona disorders kwashiorkor symptoms. Each enzyme o the urea cyce provides exampes and marasmus. Genetic disorders that resut rom deects in o metaboic deects and their physioogic consequences. In the genes that encode ubiquitin, ubiquitin igases, or deubiq- addition, the urea cyce provides a useu moecuar mode or uitinating enzymes that participate in the degradation o cer- the study o other human metaboic deects. tain proteins incude Angeman syndrome, juvenie Parkinson disease, von Hippe-Lindau syndrome, and congenita poy- cythemia. his chapter describes how the nitrogen o amino PROTEIN TURNOVER acids is converted to urea, and the metaboic disorders that he continuous degradation and synthesis (turnover) o ce- accompany deects in this process. Ammonia, which is highy uar proteins occur in a orms o ie. Each day, humans turn toxic, arises in humans primariy rom the α-amino nitrogen over 1 to 2% o their tota body protein, principay musce 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 exampe, uterine tissue during pregnancy, skeeta musce in starvation, Lysine 63 and tadpoe tai tissue during metamorphosis. Whie approxi- matey 75% o the amino acids iberated by protein degrada- tion are reutiized, the remaining excess ree amino acids are not stored or uture use. Amino acids not immediatey incor- porated into new protein are rapidy degraded. he major por- C-terminus tion o the carbon skeetons o the amino acids is converted to Lysine 48 amphiboic intermediates, whie 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 reative susceptibiity o a protein to degradation is expressed avor ubiquitination. Attachment o a singe ubiquitin moecue as its haf-ife (t1/2), the time required to ower its concentration to transmembrane proteins aters their subceuar ocaization to ha o its initia vaue. Ha-ives o iver proteins range rom and targets them or degradation. Soube proteins undergo under 30 minutes to over 150 hours. ypica “housekeeping” poyubiquitination, the igase-catayzed attachment o our or enzymes such as those o gycoysis, have t1/2 vaues o over more additiona ubiquitin moecues (Figure 28–1). Subsequent 100 hours. By contrast, key reguatory enzymes may have t1/2 degradation o ubiquitin-tagged proteins takes pace in the vaues as ow as 0.5 to 2 hours. PES sequences, regions rich in proteasome, a macromoecue that aso is ubiquitous in proine (P), gutamate (E), serine (S), and threonine (), target some proteins or rapid degradation. Intraceuar proteases hydroyze interna peptide bonds. he resuting peptides are then degraded to amino acids by endopeptidases that hydro- yze interna peptide bonds, and by aminopeptidases and carboxypeptidases that remove amino acids sequentiay rom the amino- and carboxy-termini, respectivey. ATP-Independent Degradation Degradation o bood gycoproteins (see Chapter 46) oows oss o a siaic acid moiety rom the nonreducing ends o their oigosaccharide chains. Asiaogycoproteins are then interna- ized by iver-ce asiaogycoprotein receptors and degraded by ysosoma proteases. Extraceuar, membrane-associated, and ong-ived intraceuar proteins are aso degraded in yso- somes by AP-independent processes. ATP & Ubiquitin-Dependent Degradation Degradation o reguatory proteins with short ha-ives and o abnorma or misoded proteins occurs in the cytoso, and requires AP and ubiquitin. Named based on its presence in a eukaryotic ces, ubiquitin is a sma (8.5 kDa, 76 residue) poypep- tide that targets many intraceuar proteins or degradation. he FIGURE 28–2 Reactions involved in the attachment of primary structure o ubiquitin is highy conserved. Ony 3 o 76 ubiquitin (Ub) to proteins. Three enzymes are involved. E1 is an acti- residues dier between yeast and human ubiquitin. Figure 28–1 vating enzyme, E2 a transerase, and E3 a ligase. While depicted as iustrates the three-dimensiona structure o ubiquitin. Ubiquitin single entities, there are several types o E1, and over 500 types o E2. moecues 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 transers acti- residues in the target protein (Figure 28–2). he residue present vated ubiquitin to E2. E3 then catalyzes the transer o ubiquitin to at its amino terminus aects 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 utiization by various tissues. Musce 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 cyce 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 Musce and iver thus pay major roes in maintaining circuat- degrade them to peptides. ing amino acid eves. Figure 28–5 summarizes the postabsorptive state. Free amino acids, particuary aanine and gutamine, are reeased eukaryotic ces. he proteasome consists o a macromoecuar, rom musce into the circuation. Aanine is extracted primar- cyindrica compex o proteins, whose stacked rings orm a cen- iy by the iver, and gutamine is extracted by the gut and the tra pore that harbors the active sites o proteoytic enzymes. For kidney, both o which convert a signiicant portion to aanine. degradation, a protein thus must irst enter the centra pore. Entry Gutamine aso serves as a source o ammonia or excretion into the core is reguated by the two outer rings that recognize by the kidney. he kidney provides a major source o serine poyubiquitinated proteins (Figures 28–3 and 28–4). or uptake by periphera tissues, incuding iver and musce. 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 resut rom deects in the genes NH3 that encode ubiquitin, ubiquitin igases, or deubiquitinating Brain enzymes incude Angeman syndrome, autosoma recessive juvenie Parkinson disease, von Hippe-Lindau syndrome, and congenita poycythemia. For additiona aspects o protein degradation and o ubiquitination, incuding its roe in the ce Val Ser cyce, 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 circuat- FIGURE 28–5 Interorgan amino acid exchange in normal ing pasma amino acids between meas depends on the net postabsorptive humans. The key role o alanine in amino acid out- baance between reease 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 prandiay they are extracted predominanty by musce, 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 ecoogica niche and physioogy, dier- –NH2 ent animas excrete excess nitrogen as ammonia, uric acid, or Alanine Alanine urea. he aqueous environment o teeostean ish, which are Amino acids ammonoteic (excrete ammonia), permits them to excrete Alanine water continuousy to aciitate excretion o ammonia, which is highy toxic. Whie this approach is appropriate or an aquatic anima, birds must both conserve water and maintain ow weight. Birds, which are uricoteic, address both prob- ems by excreting nitrogen-rich uric acid (see Figure 33–11) as semisoid guano. Many and animas, incuding humans, FIGURE 28–6 The glucose-alanine cycle. Alanine is synthe- are ureoteic and excrete nontoxic, highy water-soube urea. sized in muscle by transamination o glucose-derived pyruvate, Since urea is nontoxic to humans, high bood eves 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, particuary vaine, are reeased Urea biosynthesis occurs in our stages: (1) transamination, by musce and taken up predominanty by the brain. (2) oxidative deamination o gutamate, (3) ammonia trans- Aanine is a key guconeogenic amino acid (Figure 28–6). port, and (4) reactions o the urea cyce (Figure 28–8). he he rate o hepatic guconeogenesis rom aanine 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 cyce increases severaod in starvation, probaby secondary guconeogenesis rom aanine does not reach saturation unti to enhanced protein degradation to provide energy. the aanine concentration reaches 20 to 30 times its norma physioogic eve. Foowing a protein-rich mea, the spanchnic tissues reease amino acids (Figure 28–7) whie the periphera Transamination Transfers α-Amino musces extract amino acids, in both instances predominanty Nitrogen to α-Ketoglutarate, branched-chain amino acids. Branched-chain amino acids Forming Glutamate thus serve a specia roe in nitrogen metaboism. In the asting ransamination reactions interconvert pairs o α-amino acids and α-keto acids (Figure 28–9). ransamination reac- Kidney tions, which are reey reversibe, aso 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 eevated serum eves o aminotranserases (see abe 7–1). biosynthesis (see Figure 27–4). A o the common amino acids except ysine, threonine, proine, and hydroxyproine 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) readiy undergoes transamination. Aanine-pyruvate aminotranserase (aanine aminotrans- IN NITROGEN METABOLISM erase, EC 2.6.1.2) and gutamate-α-ketogutarate aminotrans- ranser o amino nitrogen to α-ketogutarate orms l-gutamate. erase (gutamate aminotranserase, EC 2.6.1.1) catayze the Hepatic l-gutamate dehydrogenase (GDH), which can use transer o amino groups to pyruvate (orming aanine) or to either NAD+ or NADP+, reeases this nitrogen as ammonia α-ketogutarate (orming gutamate). (Figure 28–12). Conversion o α-amino nitrogen to ammo- Each aminotranserase is speciic or one pair o substrates, nia by the concerted action o gutamate aminotranserase and but nonspeciic or the other pair. Since aanine is aso a sub- GDH is oten termed “transdeamination.” Liver GDH activ- strate or gutamate aminotranserase, the α-amino nitrogen ity is aostericay inhibited by AP, GP, and NADH, and is rom a amino acids that undergo transamination can be con- activated by ADP. he GDH reaction is reey reversibe, and centrated in gutamate. his is important because l-gutamate aso unctions in amino acid biosynthesis (see Figure 27–1). is the ony amino acid that undergoes oxidative deamination at an appreciabe rate in mammaian tissues. he ormation o ammonia rom α-amino groups thus occurs mainy via the AMINO ACID OXIDASES REMOVE α-amino nitrogen o l-gutamate. NITROGEN AS AMMONIA ransamination occurs via a “ping-pong” mechanism l-Amino acid oxidase o iver and kidney convert an amino characterized by the aternate addition o a substrate and acid to an α-imino acid that decomposes to an α-keto acid with reease o a product (Figure 28–10). Foowing remova o its reease o ammonium ion (Figure 28–13). he reduced avin α-amino nitrogen by transamination, the remaining carbon is reoxidized by moecuar oxygen, orming hydrogen perox- “skeeton” o an amino acid is degraded by pathways discussed ide (H2O2), which then is spit to O2 and H2O by cataase, EC in Chapter 29. 1.11.1.6. Pyridoxa phosphate (PLP), a derivative o vitamin B6, is present at the cataytic site o a aminotranserases, and pays a key roe in cataysis. 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 bood and the ammonia produced by tissues are Schi base can rearrange in various ways. In transamination, rapidy removed rom circuation by the iver and converted rearrangement orms an α-keto acid and enzyme-bound pyri- to urea. hus, normay, ony traces (10-20 μg/dL) are present doxamine phosphate. As noted earier, certain diseases are in periphera bood. 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. Shoud porta bood bypass the iver, systemic bood ammonia may reach toxic eves. his O O occurs in severey impaired hepatic unction or the deveop- L-Glutamine ment o coatera inks between the porta and systemic veins in cirrhosis. Symptoms o ammonia intoxication incude FIGURE 28–14 Formation of glutamine, catalyzed by gluta- mine synthetase, EC 6.3.1.2. tremor, surred speech, burred vision, coma, and utimatey death. Ammonia may be toxic to the brain in part because it reacts with α-ketogutarate to orm gutamate. he resuting deiciency in neonate gutamine synthetase resuts in severe depetion o α-ketogutarate then impairs unction o the tri- brain damage, mutiorgan aiure, and death. carboxyic acid (CA) cyce in neurons. Glutaminase & Asparaginase Glutamine Synthetase Fixes Ammonia Deamidate Glutamine & Asparagine as Glutamine here are two human isoorms o mitochondria gutamin- ase, termed iver-type and rena-type gutaminase. Products Formation o gutamine is catayzed by mitochondria gutamine o dierent genes, the gutaminases dier with respect to their synthetase (Figure 28–14). Since amide bond synthesis is structure, kinetics, and reguation. Hepatic gutaminase eves couped to the hydroysis o AP to ADP and Pi, the reac- rise in response to high protein intake whie rena kidney-type tion strongy avors gutamine synthesis. During cataysis, gutaminase increases in metaboic acidosis. Hydroytic reease gutamate attacks the γ-phosphory group o AP, orming o the amide nitrogen o gutamine as ammonia, catayzed by γ-gutamy phosphate and ADP. Foowing deprotonation o gutaminase (Figure 28–15), strongy avors gutamate orma- NH4+, NH3 attacks γ-gutamy phosphate, and gutamine and tion. An anaogous reaction is catayzed by l-asparaginase Pi are reeased. In addition to providing gutamine to serve (EC 3.5.1.1). he concerted action o gutamine synthetase as a carrier o nitrogen, carbon and energy between organs and gutaminase thus catayzes the interconversion o ree (Figure 28–5), gutamine synthetase pays a major roe both in ammonium ion and gutamine. ammonia detoxiication 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 utimatey become urea. he major metaboic roe o ornithine, citruine, and arginino- Maintains Acid–Base Balance succinate in mammas is urea synthesis. Urea synthesis is a cycic Excretion into urine o ammonia produced by rena tubuar process. Whie ammonium ion, CO2, AP, and aspartate are con- ces aciitates cation conservation and reguation o acid– sumed, the ornithine consumed in reaction 2 is regenerated in base baance. Ammonia production rom intraceuar rena reaction 5. hus, there is no net oss or gain o ornithine, citru- amino acids, especiay gutamine, increases in metaboic aci- ine, argininosuccinate, or arginine. As indicated in Figure 28–16, dosis and decreases in metaboic akaosis. 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 AP, 1 mo each Initiates Urea Biosynthesis o ammonium ion and o aspartate, and empoys ive enzymes Condensation o CO2, ammonia, and AP to orm carbamoy (Figure 28–16). O the six participating amino acids, phosphate is catayzed by mitochondria carbamoy phos- N-acetygutamate unctions soey as an enzyme activator. he phate synthetase I (EC 6.3.4.16). A cytosoic 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 speciic 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 gutamine aminotranserase then reorms aspartate. he carbon skeeton 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 gutamate into a precursor o urea. action o gutamate dehydrogenase and carbamoy phosphate synthetase I thus shuttes amino nitrogen into carbamoy phosphate, a compound with high group transer potentia. Cleavage of Arginine Releases Urea & Carbamoy phosphate synthetase I, the rate-imiting Reforms Ornithine enzyme o the urea cyce, is active ony in the presence o Hydroytic ceavage o the guanidino group o arginine, cata- N-acetygutamate, an aosteric activator that enhances the yzed by iver arginase (EC 3.5.3.1), reeases urea (reaction ainity o the synthetase or AP. Synthesis o 1 mo o car- 5, Figure 28–16). he other product, ornithine, reenters iver bamoy phosphate requires 2 mo o AP. One AP 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 AP provides nase, and compete with arginine. Arginine aso serves as the the driving orce or synthesis o the amide bond o carbamoy precursor o the potent musce reaxant nitric oxide (NO) in a phosphate. he other products are 2 mo o ADP and 1 mo o Ca2+-dependent reaction catayzed by NO synthetase. Pi (reaction 1, Figure 28–16). he reaction proceeds stepwise. Reaction o bicarbonate with AP orms carbony phosphate and ADP. Ammonia then dispaces ADP, orming carbamate Carbamoyl Phosphate Synthetase I and orthophosphate. Phosphoryation o carbamate by the Is the Pacemaker Enzyme of the second AP then orms carbamoy phosphate. Urea Cycle he activity o carbamoy phosphate synthetase I is determined Carbamoyl Phosphate Plus Ornithine by N-acetygutamate, whose steady-state eve is dictated by Forms Citrulline the baance between its rate o synthesis rom acety-CoA and l-Ornithine transcarbamoyase (EC 2.1.3.3) catayzes trans- gutamate and its rate o hydroysis to acetate and gutamate, er o the carbamoy group o carbamoy phosphate to orni- reactions catayzed by N-acetygutamate synthetase (NAGS) thine, orming citruine and orthophosphate (reaction 2, and N-acetygutamate deacyase (hydroase), respectivey. Figure 28–16). Whie the reaction occurs in the mitochon- Acety-CoA + l-gutamate → N-acety-l-gutamate + CoASH dria matrix, both the ormation o ornithine and the subse- N-acety-l-gutamate + H2O → l-gutamate + acetate quent metaboism o citruine take pace in the cytoso. Entry o ornithine into mitochondria and exodus o citruine rom Major changes in diet can increase the concentrations o mitochondria invoves the mitochondria inner membrane individua urea cyce enzymes 10- to 20-od. For exampe, carriers ORC1, ORC2, and SLCA25A29 (Figure 28–16). starvation eevates enzyme eves, presumaby 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 citruine via the amino group o aspartate (reaction 3, Figure 28–16), which provides the second nitrogen o urea. METABOLIC DISORDERS he reaction requires AP and invoves intermediate orma- he comparativey rare, but we-characterized and medicay tion o citruy-AMP. Subsequent dispacement o AMP by devastating metaboic disorders associated with the enzymes aspartate then orms argininosuccinate. o urea biosynthesis iustrate the oowing genera principes o inherited metaboic diseases: Cleavage of Argininosuccinate Forms 1. Simiar or identica cinica signs and symptoms can accom- Arginine & Fumarate pany various genetic mutations in a gene that encodes a given enzyme or in enzymes that catayze successive reac- Ceavage o argininosuccinate is catayzed by argininosuccinate tions in a metaboic pathway. yase (EC 4.3.2.1). he reaction proceeds with retention o a three nitrogens in arginine and reease o the aspartate 2. Rationa therapy is based on an understanding o the re- skeeton as umarate (reaction 4, Figure 28–16). Subsequent evant biochemica enzyme-catayzed reactions in both nor- addition o water to umarate orms l-maate, whose subse- ma and impaired individuas. quent NAD+-dependent oxidation orms oxaoacetate. hese 3. he identiication o intermediates and o anciary prod- two reactions are anaogous to reactions o the citric acid ucts that accumuate prior to a metaboic bock provides cyce, but are catayzed by cytosolic fumarase and maate the basis or metaboic screening tests that can impicate dehydrogenase. ransamination o oxaoacetate by gutamate the reaction that is impaired. CHAPTER 28 Catabolism o Proteins & o Amino Acid Nitrogen 287 4. Deinitive diagnosis invoves quantitative assay o the activ- suicient protein, arginine, and energy to promote growth ity o the enzyme suspected to be deective. and deveopment whie simutaneousy minimizing the meta- 5. he DNA sequence o the gene that encodes a given mutant boic perturbations. enzyme is compared to that o the wid-type gene to iden- tiy the speciic mutation(s) that cause the disease. Carbamoyl Phosphate Synthetase I 6. he exponentia increase in DNA sequencing o human N-Acetygutamate is essentia or the activity o carbamoy genes has identiied dozens o mutations o an aected phosphate synthetase I, EC 6.3.4.16 (reaction 1, Figure 28–16). gene that are benign or are associated with symptoms o Deects in carbamoy phosphate synthetase I are responsibe varying severity o a given metaboic disorder. or the reativey rare (estimated requency 1:62,000) meta- boic disease termed “hyperammonemia type 1.” METABOLIC DISORDERS ARE N-Acetylglutamate Synthetase ASSOCIATED WITH EACH N-Acetygutamate synthetase, EC 2.3.1.1 (NAGS), catayzes REACTION OF THE UREA CYCLE the ormation rom acety-CoA and gutamate o the N- Five we-documented diseases represent deects in the bio- acetygutamate essentia or carbamoy phosphate synthetase synthesis o enzymes o the urea cyce. Moecuar genetic I activity. anaysis has pinpointed the oci o mutations associated with l-Gutamate + acety-CoA → N-acety-l-gutamate + CoASH each deiciency, each o which exhibits considerabe genetic and phenotypic variabiity (Tabe 28–1). Whie the cinica and biochemica eatures o NAGS dei- Urea cyce disorders are characterized by hyperammo- ciency are indistinguishabe rom those arising rom a deect nemia, encephaopathy, and respiratory akaosis. Four o the in carbamoy phosphate synthetase I, a deiciency in NAGS ive metaboic diseases, deiciencies o carbamoy phosphate may respond to administered N-acetygutamate. synthetase I, ornithine carbamoy transerase, argininosuc- cinate synthetase, and argininosuccinate yase, resut in the Ornithine Permease accumuation o precursors o urea, principay ammonia and he hyperornithinemia, hyperammonemia, and homocitru- gutamine. Ammonia intoxication is most severe when the inuria (HHH) syndrome resuts rom mutation o the ORC1 metaboic bock occurs at reactions 1 or 2 (Figure 28–16), or gene that encodes the mitochondria membrane ornithine i citruine can be synthesized, some ammonia has aready carrier. he inabiity to import cytosoic ornithine into the been removed by being covaenty inked to an organic mitochondria matrix renders the urea cyce inoperabe, with metaboite. consequent hyperammonemia, and hyperornithinemia due Cinica symptoms common to a urea cyce disorders to the accompanying accumuation o cytosoic ornithine. In incude vomiting, avoidance o high-protein oods, intermit- the absence o its norma acceptor (ornithine), mitochondria tent ataxia, irritabiity, ethargy, and severe menta retardation. carbamoy phosphate carbamoyates ysine to homocitruine, he most dramatic cinica presentation occurs in u-term resuting in homocitruinuria. inants who initiay appear norma, then exhibit progressive ethargy, hypothermia, and apnea due to high pasma ammo- nia eves. he cinica eatures and treatment o a ive disor- Ornithine Transcarbamoylase ders are simiar. Signiicant improvement and minimization he X-chromosome–inked deiciency termed “hyperammo- o brain damage can accompany a ow-protein diet ingested nemia type 2” reects a deect in ornithine transcarbamoyase as requent sma meas to avoid sudden increases in bood (reaction 2, Figure 28–16). he mothers aso exhibit hyper- ammonia eves. he goa o dietary therapy is to provide ammonemia and an aversion to high-protein oods. Leves 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 transerase 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 gutamine are eevated in bood, cerebrospina uid, and Can Metabolic Disorders Be Rectified urine, probaby as a resut o enhanced gutamine synthesis in response to eevated eves o tissue ammonia. by Gene or Protein Modification Despite resuts in anima modes using an adenovira vector to treat citruinemia, at present gene therapy provides no eec- Argininosuccinate Synthetase tive soution or human subjects. However, direct CRISPR/ In addition to patients who ack detectabe argininosuccinate Cas9-based modiication o a deective enzyme can restore synthetase activity (reaction 3, Figure 28–16), 25-od eeva- unctiona enzyme activity o cutured human puripotent tions in Km or citruine have been reported. In the resuting stem ces. citruinemia, pasma and cerebrospina uid citruine eves are eevated, and 1 to 2 g o citruine are excreted daiy. SUMMARY Argininosuccinate Lyase Human subjects degrade 1 to 2% o their body protein daiy at rates that vary widey between proteins and with physioogic Argininosuccinic aciduria, accompanied by eevated eves state. Key reguatory enzymes oen have short ha-ives. o argininosuccinate in bood, cerebrospina uid, and urine, Proteins are degraded by both AP-dependent and AP- is associated with riabe, tuted hair (trichorrhexis nodosa). independent pathways. Ubiquitin targets many intraceuar Both eary- and ate-onset types are known. he metaboic proteins or degradation. Liver ce surace receptors bind deect is in argininosuccinate yase (reaction 4, Figure 28–16). and internaize circuating asiaogycoproteins destined or Diagnosis by the measurement o erythrocyte argininosucci- ysosoma degradation. nate yase activity can be perormed on umbiica cord bood Poyubiquitinated proteins are degraded by proteases or amniotic uid ces. on the inner surace o a cyindrica macromoecue, the proteasome. Entry into the proteasome is gated by a Arginase donut-shaped protein pore that rejects entry to a but poyubiquitinated proteins. Hyperargininemia is an autosoma recessive deect in the gene Fishes excrete highy toxic NH3 directy. Birds convert NH3 to or arginase (reaction 5, Figure 28–16). Unike other urea cyce uric acid. Higher vertebrates convert NH3 to urea. disorders, the irst symptoms o hyperargininemia typicay do ransamination channes amino acid nitrogen into not appear unti age 2 to 4 years. Bood and cerebrospina uid gutamate. GDH occupies a centra position in nitrogen eves o arginine are eevated. he urinary amino acid pattern, metaboism. which resembes that o ysine-cystinuria (see Chapter 29), Gutamine synthetase converts NH3 to nontoxic gutamine. may reect competition by arginine with ysine and cysteine Gutaminase reeases NH3 or use in urea synthesis. or reabsorption in the rena tubue. NH3, CO2, and the amide nitrogen o aspartate provide the atoms o urea. Analysis of Neonate Blood by Tandem Hepatic urea synthesis takes pace in part in the mitochondria Mass Spectrometry Can Detect matrix and in part in the cytoso. Metabolic Diseases Changes in enzyme eves and aosteric reguation o Metaboic diseases caused by the absence or unctiona impair- carbamoy phosphate synthetase I by N-acetygutamate reguate urea biosynthesis. ment o metaboic enzymes can be devastating. Eary dietary intervention, however, can in many instances ameiorate the Metaboic diseases are associated with deects in each enzyme otherwise inevitabe dire eects. he eary detection o such o the urea cyce, o the ORC1 ornithine carrier, and o NAGS. metaboic diseases is thus is o primary importance. Since Te metaboic disorders o urea biosynthesis iustrate six the initiation in the United States o newborn screening pro- genera principes o a metaboic disorders. grams in the 1960s, a states now conduct metaboic screen- andem mass spectrometry is the technique o choice or ing o newborn inants. he poweru and sensitive technique screening neonates or inherited metaboic diseases. o tandem mass spectrometry (MS) (see Chapter 4) can in a ew minutes detect over 40 anaytes o signiicance in the detection o metaboic disorders. Most states empoy tandem REFERENCES MS to screen newborns to detect metaboic disorders such as Adam S, Ameida MF, Assoun M, et a: Dietary management o urea cyce disorders: European practice. Mo Genet Metab organic acidemias, aminoacidemias, disorders o atty acid 2013;110:439. oxidation, and deects in the enzymes o the urea cyce. An Burgard P, Köker S, Haege G, et a. Neonata mortaity and outcome artice in Clinical Chemistry 2006 39:315 reviews the theory o at the end o the frst year o ie in eary onset urea cyce tandem MS, its appication to the detection o metaboic disor- disorders. J Inherit Metab Dis. 2016;39:219. ders, and situations that can yied ase positives, and incudes Dwane L, Gaagher WM, Ni Chonghaie , et a: Te emerging roe a engthy tabe o detectabe anaytes and the reevant meta- o non-traditiona ubiquitination in oncogenic pathways. J Bio boic diseases. Chem 2017;292:3543. 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