Protein and Amino Acid Metabolism

CHAPTER 17

In contrast to the case of lipids and carbohydrates, no spe- and myosin (Chapter 21) are released during catabolism cial storage forms of either the nitrogen or the amino acid of these proteins and are excreted in the urine. components of proteins exist. Dietary protein in excess Protein turnover is not completely efficient in the re- of the requirement is catabolized to provide energy and utilization of amino acids. Some are lost by oxidative ammonia, a toxic metabolite that is converted to urea in catabolism, while others are used in synthesis of non- the liver and excreted by the kidneys. All body proteins protein metabolites. For this reason, a dietary source of serve a specific function (e.g., structural, catalytic, trans- protein is needed to maintain adequate synthesis of pro- port, regulatory) and are potential sources of carbon for tein. During periods of growth, pregnancy, lactation, or energy production. recovery from illness, supplemental dietary protein is re- Proteins constantly undergo breakdown and synthesis. quired. These processes are affected by energy supply and During growth, even though there is net deposition of pro- hormonal factors. An overview of amino acid metabolism tein, the rates of synthesis and breakdown are increased. is presented in Figure 17-1. Total protein turnover in a well-fed, adult human is estimated at about 300 g/day, of which approximately 100 g is myofibrillar protein, 30 g is digestive enzymes, 20 g is 17.1 Essential and Nonessential Amino Acids small intestinal cell protein, and 15 g is hemoglobin. The remainder is accounted for by turnover of cellular pro- Plants and some bacteria synthesize all 20 amino acids teins of various other cells (e.g., hepatocytes, leukocytes, (see also Chapter 2). Humans (and other animals) can platelets) and oxidation of amino acids, and a small amount synthesize about half of them (the nonessential amino is lost as free amino acids in urine. Protein turnover rates acids) but require the other half to be supplied by the vary from tissue to tissue, and the relative tissue contri- diet (the essential amino acids). Diet must also pro- bution to total protein turnover is altered by aging, dis- vide a digestible source of nitrogen for synthesis of the ease, and changes in dietary protein intake. Several pro- nonessential amino acids. The eight essential amino acids teins (e.g., many hepatic enzymes) have short turnover are isoleucine, leucine lysine, methionine, phenylalanine, times (less than 1 hour), whereas others have much longer threonine, tryptophan, and valine. In infants, histidine (and turnover times (e.g., collagen > 1000 days). Turnover of possibly arginine) is required for optimal development myofibrillar protein can be estimated by measurement of and growth and is thus essential. In adults, histidine is 3-methylhistidine in the urine. Histidyl residues of actin nonessential, except in uremia. Under certain conditions,

331 332 CHAPTER 17 Protein and Amino Acid Metabolism

balance exists when the amount of nitrogen lost from the body (as nitrogen metabolites excreted in urine and feces) exceeds that taken in. This state continues until the essential amino acid deficiency is corrected. Negative nitrogen balance also occurs in malabsorption syndromes, fever, trauma, cancer, and excessive production of catabolic hormones (e.g., hypercortisolism; see Chapter 32). When the dietary nitrogen intake equals nitrogen losses, the body is in nitrogen balance. In normal adults, anabolism equals catabolism. When nitrogen intake exceeds nitrogen losses, there is a positive nitrogen balance, with anabolism ex- ceeding catabolism. The body retains nitrogen as tissue protein, which is a characteristic of active growth and tissue repair (e.g., growth in children, pregnancy, recovery from an emaciating illness).

Quality and Quantity of Dietary Protein Requirement FIGURE 17-1 Dietary protein provides organic nitrogen and the essen- Overall metabolism of proteins and amino acids. Body protein is maintained by the balance between the rates of protein synthesis and tial amino acids. The quantitative estimation of protein breakdown. These processes are influenced by hormones and energy requirement must take into account the quality of protein, supply. as determined by its essential amino acid composition. Dietary protein should provide all of the essential amino acids in the appropriate amounts. If the concentration of some nonessential amino acids may become essential. For one amino acid is significantly greater or less than that of example, when liver function is compromised by disease the others (in a protein or amino acid mixture), utilization or premature birth, cysteine and tyrosine become essential of the others may be depressed and will be reflected in because they cannot be formed from their usual precursors growth failure. (methionine and phenylalanine). A procedure for assessment of protein quality consists Glutamine, a nitrogen donor in the synthesis of purines of feeding growing rats various levels of the test protein and pyrimidines required for nucleic acid synthesis (Chap- and assessing the slope of regression lines relating growth ter 27), aids in growth, repair of tissues, and promotion rate and protein intake. Wheat protein is deficient in ly- of immune function. Enrichment of glutamine in enteral sine when compared with lactalbumin, which contains all and parenteral nutrition augments recovery of seriously of the essential amino acids in desirable concentrations. ill patients. Arginine may be considered as a semiessen- Wheat protein is therefore assessed to be 20% as effec- tial amino acid. It participates in a number of metabolic tive as an equivalent amount of lactalbumin. Similarly, pathways, namely, formation of urea and ornithine, crea- proteins from corn, which are also deficient in lysine, do tine and creatinine, spermine, agmatine and citrulline, and not support optimal growth. However, genetic selection nitric oxide (NO). The endothelial cells lining the blood and breeding programs have yielded strains of corn with vessels produce NO from arginine, which has a major role higher lysine content. Proteins of animal originmnamely, in vasodilator function (discussed later). Dietary arginine meats, eggs, milk, cheese, poultry, and fishmare of good supplementation improves coronary blood flow, reduces quality since they provide all of the essential amino acids. episodes of angina, and helps in patients with walking Gelatin, the protein derived from collagen, lacks trypto- pain due to claudication. phan and is of poor quality. In general, plant proteins are of poor quality because they lack one or more essential amino acids. The best quality plant proteins are found in Nitrogen Balance legumes and nuts. Therefore, the diet of a pure vegetarian For protein synthesis to occur, all 20 amino acids must requires careful planning to achieve a combination of pro- be present in sufficient quantities. Absence of any one teins that provide necessary amounts of all essential amino essential amino acid leads to cessation of protein synthe- acids. Combinations of complementary vegetable proteins sis, catabolism of unused amino acids, increased loss of include rice and black-eyed peas; whole wheat or parched nitrogen in urine, and reduced growth. Negative nitrogen crushed wheat (bulgur) with soybeans and sesame seeds; SECTION 17.1 Essential and Nonessential Amino Acids 333 cornmeal and kidney beans; and soybeans, peanuts, brown stay in the hospital. Thus, protein energy malnutrition can rice, and bulgur (Chapter 12). Deficiency of vitamin B12 cause morbidity, mortality and also has economic con- (Chapter 38) may occur in persons on a pure vegetarian sequences. Acute stressful physiological conditions such diet. Although plant proteins used singly do not provide as trauma, burn, or sepsis can also precipitate protein en- all of the essential amino acids, their inclusion in the diet ergy malnutrition due to hypermetabolism caused by the provides nonessential amino acids that would otherwise neuroendocrine system. Prompt diagnosis and appropriate have to be synthesized at the expense of the nitrogen of nutritional intervention is required in the management of the essential amino acids. The following estimates of daily patients with protein energy malnutrition. protein needs are for persons on a Western diet: adults, Measurements of the levels of serum proteins such as al- 0.8 g/kg; newborns, 2.2 g/kg; infants (0.5-1 year), 2 g/kg. bumin, transthyretin (also known as prealbumin), transfer- During pregnancy and lactation protein intake above the rin and retinol-binding protein are used as biochemical pa- normal adult level is recommended (Appendix IV). These rameters in the assessment of protein energy malnutrition protein requirements are valid only when energy needs (Table 17-1). An ideal protein marker should have rapid are adequately met from nonprotein sources. If intake of turnover and present in sufficiently high concentrations in carbohydrates and lipids is insufficient to meet the energy serum to be measured accurately. Transthyretin has these expenditure, dietary protein is utilized to meet the energy properties; it is a sensitive indicator of protein deficiency deficit and results in negative nitrogen balance. and is effective in assessing improvement with refeeding.

Protein Energy Malnutrition Transport of Amino Acids into Cells Two disorders of protein energy nutrition that are wide- Intracellular metabolism of amino acids requires their spread among children in economically depressed areas transport across the cell membrane. Transport of L-amino are kwashiorkor (in Ghana, "the disease the first child gets acids occurs against a concentration gradient and is an ac- when the second is on the way") and marasmus (from the tive process usually coupled to Na+-dependent carrier sys- Greek "to waste away"). tems as for transport of glucose across the intestinal mu- Kwashiorkor occurs after weaning and is due to inade- cosa (Chapter 12). At least five transport systems for amino quate intake of good-quality protein and a diet consisting acids (with overlapping specificities) have been identified primarily of high-starch foods (e.g., yams, potatoes, ba- in kidney and intestine. They transport neutral amino acids, nanas, maize, cassava) and deficient in other essential nu- acidic amino acids, basic amino acids, ornithine and cys- trients. Victims have decreased mass and function of heart tine, and glycine and proline, respectively. Within a given and kidneys; decreased blood volume, hematocrit, and carrier system, amino acids may compete for transport serum albumin concentration; atrophy of pancreas and in- (e.g., phenylalanine with tryptophan). Na+-independent testines; decreased immunological resistance; slow wound transport carriers for neutral and lipophilic amino acids healing; and abnormal temperature regulation. Character- have also been described. D-Amino acids are transported istic clinical signs include edema, ascites, growth failure, by simple diffusion favored by a concentration gradient. apathy, skin rash, desquamation, pigment changes and Inherited defects in amino acid transport affect epithe- ulceration, loss of hair, liver enlargement, anorexia, and lial cells of the gastrointestinal tract and renal tubules. diarrhea. Some affect transport of neutral amino acids (Hartnup Marasmus results from deficiency of protein and energy disease), others that of basic amino acids and ornithine intake, as in starvation, and results in generalized wasting and cystine (r or of glycine and proline (Chap- (atrophy of muscles and subcutaneous tissues, emaciation, ter 12). Cystinosis is an intracellular transport defect char- loss of adipose tissue) Edema occurs in kwashiorkor but acterized by high intralysosomal content of free cystine in not in marasmus; however, the distinction between these the reticuloendothelial system, bone marrow, kidney, and disorders is not always clear. The treatment of marasmus eye. After degradation of endocytosed protein to amino requires supplementation of protein and energy intake. acids within lysosomes, the amino acids normally are Protein energy malnutrition occurs with high frequency transported to the cytosol. The defect in cystinosis may (30%-50%) in hospitalized patients as well as in pop- reside in the ATP-dependent efflux system for cystine ulations in chronic care facilities as either an acute or transport, and particularly in the carrier protein. a chronic problem. These individuals suffer from inad- A different mechanism for translocation of amino equate nutrition due to a disease or depression, and are acids in some cells is employed in the v-glutamyl cycle susceptible to infections due to impaired immune func- (Figure 17.2). Its operation requires six enzymes (one tion. Surgical patients with protein energy malnutrition membrane-bound, the rest cytosolic), glutathione (GSH; exhibit delayed wound healing with increased length of v-glutamylcysteinylglycine present in all tissue cells), {.} ,~

0 op~ o m op~

m c~ ~o omIN 0

mmU

0 0 om~

{D ~ ~o

ooo'

ir~ 0 0 I ~ V " r~ s.

{,-q oo I I I o it} ~ 0 oo

o 9~ ~o o o 0 ~~o ~ 0 0 0 t,, {D

~ oo ~ o~

ct~ T~ 8~

o'~ ~ ,-~ ~{}

E ~ ~ 0 .~ {} ,.I::} ~o ~o # SECTION 17.1 Essential and Nonessential Amino Acids 335

OUTSIDE MEMBRANE INSIDE Measurement of serum v-GT activity has clinical signi-

7-Glutamyl-amino acid ficance. The enzyme is present in all tissues, but the highest "",~-Glutamylcyclotransferase level is in the kidney; however, the serum enzyme orig- / "~1 Amino~cidl inates primarily from the hepatobiliary system. Elevated 5-Oxbprol~e (f~ Cysteinylglycine ~(~5.~0TPprolinase levels ofserum y-GT are found in the following disorders: IAmino acid]-- fT-Glutamy~ kHO 2H=O FADP + p~ ~ransferasej ~ ~DipeptidaseGlutamate intra- and posthepatic biliary obstruction (elevated serum \ [~ Cysteine-~.-- ATP v-GT indicates cholestasis, as do leucine aminopeptidase, \ Glycine /~7-GC synthetase x ~, J ~"ADP + P, 5'-nucleotidase, and alkaline phosphatase); primary or GS~.~~~ ~ta myIcystein e disseminated neoplasms; some pancreatic cancers, especially when associated with hepatobiliary obstruc- ADP + P, ATP tion; alcohol-induced liver disease (serum v-GT may be FIGURE 17-2 exquisitely sensitive to alcohol-induced liver injury); and The F-glutamyl cycle for the transport of amino acids. GSH = some prostatic carcinomas (serum from normal males has Glutathione; F-GC = y-glutamylcysteine. 50% higher activity than that of females). Increased activity is also found in patients receiving phenobarbital or and ATP (three ATP molecules are required for each phenytoin, possibly due to induction of v-GT in liver cells amino acid translocation). In this cycle, there is no net by these drugs. consumption of GSH, but an amino acid is transported at the expense of the energy of peptide bonds of GSH, which has to be resynthesized. Translocation is initi- General Reactions of Amino Acids ated by membrane-bound y-glutamyltransferase (y-GT, Some general reactions that involve degradation or in- y-glutamyl transpeptidase), which catalyzes formation of terconversion of amino acids provide for the synthesis a y-glutamyl amino acid and cysteinylglycine. The lat- of nonessential amino acids from o~-keto acid precursors ter is hydrolyzed by dipeptidase to cysteine and glycine, derived from carbohydrate intermediates. which are utilized in resynthesis of GSH. The former is cleaved to the amino acid and 5-oxoproline (pyroglutamic Deamination acid) by y-glutamylcyclotransferase. The cycle is completed by conversion of 5-oxoproline to glutamate and by Removal of the o~-amino group is the first step in resynthesis of GSH by two ATP-dependent enzymes, Y- catabolism of amino acids. It may be accomplished ox- glutamylcysteine synthetase and glutathione synthetase, idatively or nonoxidatively. respectively. GSH synthesis appears to be regulated by Oxidative deamination is stereospecific and is cat- nonallosteric inhibition of y-glutamylcysteine synthetase. alyzed by L- or D-amino acid oxidase. The initial step is GSH has several well-established functions: it provides removal of two hydrogen atoms by the flavin coenzyme, reducing equivalents to maintain-SH groups in other with formation of an unstable o~-amino acid intermediate. molecules (e.g., hemoglobin, membrane proteins; Chap- This intermediate undergoes decomposition by addition ter 15); it participates in inactivation of hydrogen perox- of water and forms ammonium ion and the correspond- ide, other peroxides, and free radicals (Chapter 14); it par- ing ot-keto acid: L-Amino acid oxidase occurs in the liver ticipates in other metabolic pathways (e.g., leukotrienes; H Chapter 18); and it functions in inactivation of a variety L-Amino acid I oxidase of foreign compounds by conjugation through its sulfur R--C--COO- atom. The conjugation reaction is catalyzed by specific t f-'x glutathione S-transferases and the product is eventually N H 3 + Flavin Flavin-H2 converted to mercapturic acids and excreted. ~-Amino Inherited deficiency of GSH synthetase, y-glutamy- acid lcysteine synthetase y-glutamyltransferase, and 5-oxopro- H202 02 linase have been reported. Red blood cells, the central nervous system, and muscle may be affected. In GSH and kidney only. It is a flavoprotein that contains flavin synthetase deficiency, y-glutamylcysteine accumulates mononucleotide (FMN) as a prosthetic group and does not from lack of inhibition of y-glutamylcysteine synthetase attack glycine, dicarboxylic, or fi-hydroxy amino acids. Its by glutathione, and it is converted to 5-oxoproline and activity is very low. cysteine by y-glutamylcyclotransferase. 5-Oxoproline High levels of D-amino acid oxidases are found in the is excreted so that GSH synthetase deficiency causes liver and kidney. The enzyme contains flavin adenine din- 5-oxoprolinuria (or pyroglutamic aciduria). ucleotide (FAD) and deaminates many D-amino acids and 336 CHAPTER 17 Protein and Amino Acid Metabolism glycine. The reaction for glycine is analogous to that for Nonoxidative deamination is accomplished by several D-amino acids. specific enzymes. Amino acid dehydratases deaminate hydroxyamino H acids: D-Aminoacid l oxidase H3N__C__CO O- Seri ne ) Specific H20 I FAD FADH2 dehydratase H Threonine Homoseri ne (Pyridoxai phosphate) Glycine H202 02 NH3 0t-lmino ~" ; 0t-Keto acid H20 acid H H20 I x.. * H2N--C--CO O - 0t-lmino acid For serine dehydratase the reaction is NH4 + H20 H HO--CH2--CH--COO- ~)i 'H2C=C--COO- I I I O------C--COO- NH3 + NH3 + Glyoxylate Serine D-Amino acid oxidase occurs in peroxisomes containing other enzymes that produce H202 (e.g., L-c~-hydroxy II H20 1 acid oxidase, citrate dehydrogenase, and L-amino acid ox- H3C--C--COO-~ H3C--C--COO- Pyruvate NH3 II idase) and catalase and peroxidase, which destroy H202. NH2 + In leukocytes, killing of bacteria involves hydrolases of lysosomes and production of H202 by NADPH oxidase (Chapter 15). Conversion of D-amino acids to the corre- Dehydrogenation of L-Glutamate sponding o~-keto acids removes the asymmetry at the ct- carbon atom. The keto acids may be aminated to L-amino Glutamate dehydrogenase plays a major role in acids. By this conversion from D- to L-amino acids, the amino acid metabolism. It is a zinc protein, requires body utilizes D-amino acids derived from the diet: NAD + or NADP + as coenzyme, and is present in high concentrations in mitochondria of liver, heart, muscle, and R kidney. It catalyzes the (reversible) oxidative deamination D-Amino acid I oxidase of L-glutamate to ct-ketoglutarate and NH3. The initial H--C--NH3 + step probably involves formation of ot-iminoglutarate by I dehydrogenation. This step is followed by hydrolysis of COO - the imino acid to a keto acid and NH3: D-Amino acid (not useful for protein H synthesis) I -OOC--CH2--CH2--C--COO- + NAD(P) + R I Reamination NH3 + I (lransaminase) C--O L-Glutamate I CO0 - -OOC--CH2--CH2--C--COO- + NAD(P)H + H + II 0t-Keto acid (has no NH2 + asymmetric 0t-carbon) 0t-lminoglutaric acid R I H20 ~~ H20 +H3N--C--H I COO- - OOC--CH2--CH2--C--COO- + NH4 + II L-Amino acid O (metabolically useful) ~x-Ketoglutarate SECTION 17.1 Essential and Nonessential Amino Acids 337

Glutamate dehydrogenase is an allosteric protein mod- L-Glutamate =-Ketoglutarate Glutamate-pyruvate ulated positively by ADP, GDP, and some amino acids and transaminase (GPT) or alanine aminotransferase (ALT) negatively by ATE GTP, and NADH. Its activity is affected + ( ) + by thyroxine and some steroid hormones in vitro. Gluta- mate dehydrogenase is the only amino acid dehydroge- Pyruvate L-Alanine nase present in most cells. It participates with appropriate L-Glutamate =-Ketoglutarate Glutamate-pyruvate transaminases (aminotransferases) in the deamination of transaminase (GPT) or alanine arninotransferase (ALT) other amino acids. + ( ) +

R~CH~COO- *--,.,,. /.,-."~ =-Ketoglutarate NAD(P)H + NH4 + Oxaloacetate L-Aspartate ~NH3+ Y L-Amino acid ~ All of the amino acids except lysine, threonine, proline, R--~ mCOO-~ I ~ L-Glutamate NAD(P) + + H20 and hydroxyproline participate in transamination reac- u tions. Transaminases exist for histidine, serine, pheny- I =-Keto acid Transaminase Glutamate lalanine, and methionine, but the major pathways of (aminotra n sferase) dehyd rogen ase pyridoxal phosphate their metabolism do not involve transamination. Transam- enzyme ination of an amino group not at the or-position can These reactions are at near-equilibrium, so their over- also occur. Thus, transfer of 6-amino group of ornithine all effect depends upon concentrations of the substrates to o~-ketoglutarate converts ornithine to glutamate-v- and products. Aminotransferases occur in cytosol and mi- semialdehyde. tochondria, but their activity is much higher in cytosol. All transaminase reactions have the same mechanism Since glutamate dehydrogenase is restricted to mitochon- and use pyridoxal phosphate (a derivative of vitamin B6; dria, transport of glutamate (generated by various transam- Chapter 38). Pyridoxal phosphate is linked to the enzyme inases) into mitochondria by a specific carrier becomes of by formation of a Schiff base between its aldehyde group central importance in amino acid metabolism. The NH3 and the e-amino group of a specific lysyl residue at the produced in deamination reactions must be detoxified by active site and held noncovalently through its positively conversion to glutamine and asparagine or to urea, which charged nitrogen atom and the negatively charged phos- is excreted in urine. The NADH generated is ultimately phate group (Figure 17-3). During catalysis, the amino oxidized by the electron transport chain. acid substrate displaces the lysyl e-amino group of the enzyme in the Schiff base. An electron pair is removed from the or-carbon of the substrate and transferred to the posi- Transamination tively charged pyridine ring but is subsequently returned Transamination reactions combine reversible amination and deamination, and they mediate redistribution of amino groups among amino acids. Transaminases (aminotransferases) are widely distributed in human tissues and are particularly active in heart muscle, liver, skeletal muscle, and kidney. The general reaction of transamination is ~ N COO - COO - COO - COO - HO~ ~ ~CH2~ 0 \ "C---O \ I I T,a.sa~i~ I I HC~NH3 + + C--O i ~ C---O + HC~NH3 + - 1 I I (pyrid~ ph~ I I T H RI R2 R~ R2

Amino acid Keto acid Keto acid Amino acid (1) (2) (1) (2)

The ot-ketoglutarate/L-glutamate couple serves as an amino group acceptor/donor pair in transaminase reactions. The specificity of a particular transaminase is for FIGURE 17-3 the amino group other than the glutamate. Two transami- Binding of pyridoxal phosphate to its apoenzyme. The carbonyl carbon reacts with the e-amino group of the lysyl residue near the active site to nases whose activities in serum are indices of liver damage yield a Schiff base. Ionic interactions involve its positively charged catalyze the following reactions: pyridinium ion and negatively charged phosphate group. 338 CHAPTER17 Protein and Amino Acid Metabolism

R--CH--COO- I H NH2 I (z-Amino acid R--C--COO- R--C--COO- ! Ii .N ,N CHO H,.,.'. II OH."1 H( CH2"--O--(~ H,O 0 H+ 0

-- 3, _ 3 , H~C--'~.- -~ H H H Pyridoxal phosphate Aldimine Ketimine H,O, oH" I R__ ICI__COO- or acid ~H2 CH2 Hl~~t./CH;"" O--(~

H~C H

Pyridoxamine phosphate FIGURE 17-4 Mechanism of the first phase of transamination. The -NH2 group from the amino acid is transferred to pyridoxal phosphate, with formation of the corresponding ot-keto acid. The second phase occurs by the reversal of the first phase reactions and is initiated by formation of a Schiff base with the c~-keto acid substrate and pyridoxamine phosphate. The transamination cycle is completed with formation of the corresponding or-amino acid and pyridoxal phosphate. to the second substrate, the c~-keto acid. Thus, pyridoxal Role of Specific Tissues in Amino Acid Metabolism phosphate functions as a carrier of amino groups and as an The specific roles of various tissues and organs and their electron sink by facilitating dissociation of the a-hydrogen interdependence on amino acid metabolism are discussed of the amino acid (Figure 17-4). In the overall reaction, the here. An overview of this topic is given in Chapter 22. amino acid transfers its amino group to pyridoxal phos- In the post absorptive state, maintenance of steady-state phate and then to the keto acid through formation of pyri- concentrations of plasma amino acids depends on release doxamine phosphate as intermediate. Pyridoxal phosphate is also the prosthetic group of amino acid decarboxylases, dehydratases, desulfhydrases, Bond labilizedby racemases, and aldolases, in which it participates through H O transaminases its ability to render labile various bonds of an amino acid molecule (Figure 17-5). Several drugs (Figure 17-6) in- on0 T '< ,.b,,,zo0 b, hibit pyridoxal phosphate-dependent enzymes. Isonico- by aldolases H~'~N""Ht., deca~oxylases tinic hydrazide (used in the treatment of tuberculosis) and hydralazine (a hypertensive agent) react with the aldehyde group of pyridoxal (free or bound) to form pyridoxal hydrazones, which are eliminated in the urine. Isonico- L,%.~,/,#---CH, tinic acid hydrazide is normally inactivated in the liver I by acetylation; some individuals are "slow acetylators" H (an inherited trait) and may be susceptible to pyridoxal FIGURE 17-5 deficiency from accumulation of the drug. Cycloserine Labilization of bonds of an amino acid bound to pyridoxal phosphate-containing enzymes. Given the appropriate apoenzyme, any (an amino acid analogue and broad-spectrum antibiotic) atom or group on the carbon atom proximal to the Schiff base can be also combines with pyridoxal phosphate. cleaved. SECTION 17.1 Essential and Nonessential Amino Acids 339

NH2 COO- I NH NH2 l I I (CH2)2 C:O NH I + ATP 4 + NH4 + CHNH3 + I U2X coo - H,N L-Glutamate Isonicotinic acid hydrazide Hydralazine Cycloserine (antituberculosis drug) (hypertensiveagent) (antibiotic) CONH2 FIGURE 17-6 I Structures of compounds that inhibit pyridoxal phosphate-containing (CH2)2 enzymes. I + ADP 3- + Pi 2- 4- H + CHNHa + I of amino acids from tissue protein. After a meal, dietary coo - amino acids enter the plasma and replenish the tissues that L-Glutamine supply amino acid during fasting. Liver plays a major role, since it can oxidize all amino acids except leucine, isoleucine, and valine (see Chap- Glutamate is derived by transamination of oe-keto- ter 22). It also produces the nonessential amino acids from glutarate produced in the TCA cycle from citrate via ox- the appropriate carbon precursors. Ammonia formed in the aloacetate and acetyl-CoA (Chapter 13). All of the amino gastrointestinal tract or from various deaminations in the acids can produce acetyl-CoA. All except leucine and ly- liver is converted to urea and excreted in urine (discussed sine (which are oxidized solely to acetyl-CoA) can be used later). in net synthesis of ot-ketoglutarate to enhance glutamate Skeletal muscle tissue constitutes a large portion of the synthesis. Ammonia is generated in glutamate dehydro- body weight and accounts for a significant portion of non- genase and AMP deaminase reactions (Chapter 21). hepatic amino acid metabolism. It takes up the amino acids The mucosa of the small intestine metabolizes dietary required to meet its needs for protein synthesis, and me- glutamine, glutamate, asparagine, and aspartate by oxida- tabolizes alanine, aspartate, glutamate, and the branched- tion to CO2 and H20, or by conversion to lactate, alanine, chain amino acids. Amino acids are released from muscle citrulline, and NH3. These intermediates and the unme- during the postabsorptive state (i.e., in fasting or starva- tabolized dietary amino acids are transferred to the portal tion). Alanine and glutamine constitute more than 50% of blood and then to the liver for further metabolism. the or-amino acid nitrogen released. During starvation, the In the fasting state, the intestinal mucosa depends on total amino acid pool increases from catabolism of con- other tissues for metabolites to provide energy and precur- tractile proteins. However, the amino acid composition of sors for protein and nucleotide synthesis to maintain the these proteins does not account for the large amount of ala- rapid cell division characteristic of that tissue. Glutamine, nine and glutamine released. Amino acids that give rise to released from liver and muscle, is utilized for purine pyruvate can be transaminated to alanine. For example, nucleotide synthesis (Chapter 27), is oxidized to provide aspartate can be converted to alanine as follows: energy, and can be converted to aspartate for pyrimidine nucleotide synthesis (Chapter 27). Thus, glutamine is im- transaminase PEPCK Aspartate > Oxaloacetate > portant in cells undergoing rapid division. Intestine can pyruvate kinase also oxidize glucose, fatty acids, and ketone bodies to Phosphoenolpyruvate > provide energy. transaminase Kidney releases serine and small (but significant) quan- Pyruvate > Alanine tities of alanine into the blood, and takes up glutamine, Similarly, amino acids that produce tricarboxylic acid proline, and glycine. Amino acids filtered in the glomeruli (TCA) cycle intermediates (Chapter 15) produce alanine are reabsorbed by renal tubule cells. Glutamine plays an by conversion to oxaloacetate. During starvation or in- important role in acid-base regulation by providing am- take of a carbohydrate-poor diet, conversion of pyruvate monia, which forms the NH~- ion eliminated in urine to alanine is preferred because pyruvate dehydrogenase is (Chapter 39). It can provide two ammonia molecules, by inactivated by oxidation of fatty acids and ketone bodies glutaminase and glutamate dehydrogenase, respectively, (Chapters 13 and 18). in renal tubular mitochondria. Its carbon skeleton can Glutamine is synthesized from glutamate and ammonia be oxidized or converted to glucose since renal tissue is by glutamine synthase: capable of gluconeogenesis (Chapter 15). 340 CHAPTER 17 Protein and Amino Acid Metabolism

Brain takes up significant quantities of valine and Ammonia is particularly toxic to brain but not to may be a major (if not primary) site of utilization of other tissues, even though levels in those tissues may branched-chain amino acids. Glutamate, aspartate, and increase under normal physiological conditions (e.g., in glycine are neurotransmitters. Glutamate is a precursor of muscle during heavy exercise in kidney during metabolic y-aminobutyrate; tyrosine of dopamine, norepinephrine, acidosis). Several hypotheses have been suggested to and epinephrine; and tryptophan of serotonin, all of which explain the mechanism of neurotoxicity. are neurotransmitters. Inactivation of neurotransmitters in- In brain mitochondria, excess ammonia may drive the volves deamination with production of ammonia, which is reductive amination of ot-ketoglutarate by glutamate de- removed by formation of glutamine. N-acetylaspartate oc- hydrogenase. This step may deplete a key intermedi- curs in high levels in the brain but its function is not known. ate of the TCA cycle and lead to its impairment, with It is synthesized from acetyl-CoA and aspartic acid cat- severe inhibition of respiration and considerable stim- alyzed by acetyl-CoA aspartate N-acetyl transferase. As- ulation of glycolysis. Since the [NAD+]/[NADH] ratio partoacylase catalyzes the hydrolysis of N-acetylaspartate will be high in mitochondria, there will be a decrease to acetate and aspartic acid. The deficiency of aspartoa- in the rate of production of ATE This hypothesis does cylase, which is inherited as an autosomal recessive trait, not explain why the same result does not occur in tis- is associated with degenerative brain changes. Patients of sues that are not affected by ammonia. A more plau- this disorder, also known as Canavan dystrophy, are usu- sible hypothesis is depletion of glutamate which is an ally of Eastern European Jewish heritage. excitatory neurotransmitter. Glutamine, synthesized and stored in glial cells, is the most likely precursor of glutamate. It is transported into the neurons and hydrolyzed 17.2 Metabolism of Ammonia by glutaminase. Ammonia inhibits glutaminase and de- pletes the glutamate concentration. A third hypothesis Ammonia (at physiological pH, 98.5% exists as NH+), the invokes neuronal membrane dysfunction, since elevated highly toxic product of protein catabolism, is rapidly in- levels of ammonia produce increased permeability to K + activated by a variety of reactions. Some products of these and C1- ions, while glycolysis increases H + ion concen- reactions are utilized for other purposes (thus salvaging a tration (NH~- stimulates 6-phosphofructokinase; Chap- portion of the amino nitrogen), while others are excreted. ter 13). Encephalopathy of hyperammonemia is charac- The excreted form varies quite widely among vertebrate terized by brain edema and astrocyte swelling. Edema and and invertebrate animals. The development of a pathway swelling have been attributed to intracellular accumulation for nitrogen disposal in a species appears to depend chiefly of glutamine which causes osmotic shifts of water into the on the availability of water. Thus, urea is excreted in cell. terrestrial vertebrates (ureotelic organisms); ammonia in Behavioral disorders such as anorexia, sleep distur- aquatic animals (ammonotelic organisms); and uric acid bances, and pain insensitivity associated with hyperam- (in semisolid form) in birds and land-dwelling reptiles monemia have been attributed to increased tryptophan (uricotelic organisms). During their aquatic phase of transport across the blood-brain barrier and the accumu- development amphibia excrete ammonia but the adult frog lation of its metabolites. Two of the tryptophan-derived excretes urea; during metamorphosis the liver produces the metabolites are serotonin and quinolinic acid (discussed enzymes required for their synthesis. In humans, ammonia later). The latter is an excitotoxin at the N-methyl-D- is excreted mostly as urea, which is highly water-soluble, aspartate (NMDA) glutamate receptors. Thus, the mecha- is distributed throughout extracellular and intracellular nism of the ammonium-induced neurological abnormali- body water, is nontoxic and metabolically inert, has a high ties is multifactorial. Normally only small amounts of NH3 nitrogen content (47%), and is excreted via the kidneys. (i.e., NH +) are present in plasma, since NH3 is rapidly re- Ammonia is produced by deamination of glutamine, moved by reactions in tissues of glutamate dehydrogenase, glutamate, other amino acids, and adenylate. A consid- glutamine synthase, and urea formation. erable quantity is derived from intestinal bacterial enzymes acting on urea and other nitrogenous compounds. Urea Synthesis The urea comes from body fluids that diffuse into the intestine, and the other nitrogenous products are derived Ammonia contained in the blood flowing through the hep- from intestinal metabolism (e.g., glutamine) and ingested atic lobule is removed by the hepatocytes and converted protein. The ammonia diffuses across the intestinal mu- into urea. Periportal hepatocytes are the predominant sites cosa to the portal blood and is converted to urea in the of urea formation. Any ammonia that is not converted liver. to urea may be incorporated into glutamine catalyzed SECTION 17.2 Metabolism of Ammonia 341

NH+ 2ATP 2ADP + P~ Ls . Carbamoyl~ CPSI phosphate CO2 +

N-Acetylglutam ate Ornithine Citrulline C~ s

AcetyI-CoA + Glutamate

Mitochondrial inner membrane

Mitochondrial outer membrane

Ornithine/ lCitrulline UreaA~ H.O 1 ATP~ As Aspartate AMP +PP~/)

Arginine Argininosuccinate

Fumarate FIGURE 17-7 Formation of urea in hepatocytes. NAGS = N-acetylglutamate synthase; CPSI = carbamoylphosphate synthase I; OCT -- ornithine carbamoyltransferase; C-OT = citrulline-ornithine translocase; AS = argininosuccinate synthase; AL = argininosuccinate lyase; A = arginase. --Q-+ indicates the absolute requirement of N-acetylglutamate for CPSI activity. by glutamine synthase located in pericentral hepatocytes. of CoA derivatives that are also competitive inhibitors Formation of urea requires the combined action of two of N-acetylglutamate synthase and inhibitors of CPSI. enzymes to produce carbamoyl phosphate and of four Hyperammonemia often accompanies organic acidemias. enzymes that function in a cyclic manner in the urea cycle (Figure 17-7). Although some of these enzymes COO- COO- occur in extrahepatic tissues and urea formation has been l A,g I O (CH2)2 i (CH2)2 shown to occur in several cell lines in tissue culture, II I i~I I the most important physiological site of urea formation CH3C~SCoA+ CHNH 3+ =~iv ;. CoASH + CHNHCOCH3 + H + is the liver. In hepatocytes the first three enzymes are I ', I , COO mitochondrial and the others are cytosolic. A citrulline- CO0 - ' " e ornithine antiport is located in the inner mitochondrial AcetyI-CoA Glutamate "" -- -- N-Acetyl- membrane. glutamate

CPSI catalyzes the reaction Formation of Carbamoyl Phosphate Carbamoyl phosphate synthesis requires amino acid NH4 + + HCO3- 4- 2ATP4- NAG'K+'Mg2+ acetyltransferase (N-acetylglutamate synthase, mitochon- O drial) and carbamoyl-phosphate synthase I (CPSI). N- I! Acetylglutamate (NAG) is an obligatory positive effector H2N--C--OPO3 2- + 2ADP3- + Pi 2- + 2H + of CPSI. NAG synthase is under positive allosteric mod- ulation by arginine and product inhibition by NAG. De- NAG binding changes the conformation and subunit pletion of CoA-SH decreases NAG synthesis and ureage- structure of CPSI, with preponderance of the monomers. nesis. This situation can occur in organic acidemias (e.g., Carbamoylglutamate is also an activator of CPSI. Glu- propionic acidemia; Chapter 18), in which organic acids tamate and ot-ketoglutarate compete with NAG for bind- produced in excess compete for CoA-SH for formation ing. CPSI is subject to product inhibition by Mg-ADE It 342 CHAPTER 17 Protein and Amino Acid Metabolism possesses two binding sites for ATE One ATP is utilized is transported out of the mitochondria by the citrulline- in activation of bicarbonate by forming an enzyme-bound ornithine antiporter. carboxyphosphate that reacts with ammonium ion to form an enzyme-bound carbamate, with elimination of inorganic phosphate. Carbamoyl phosphate is generated when the second ATP reacts with the enzyme-bound carbamate, Formation of Argininosuccinate with release of ADP and free enzyme. The condensation of citrulline and aspartate to argini- In humans, there are two immunologically distinct car- nosuccinate is catalyzed by argininosuccinate synthase in bamoyl phosphate synthases, one mitochondrial (CPSI) the cytosol and occurs in two steps. In the initial step, and the other cytosolic (CPSII). CPSI is involved in ure- the ureido group is activated by ATP to form the enzyme- agenesis, uses NH3 exclusively as the nitrogen donor, bound intermediate adenylylcitrulline. In the second step, and requires binding of NAG for activity. CPSII uses nucleophilic attack of the amino group of aspartate glutamine as substrate, is not dependent on NAG for displaces AMP and yields argininosuccinate. The overall activity, and is required for synthesis of pyrimidine reaction is shown below:

NH2 NH COO- I I I C---O ~NH+--CH I I I NH COO- NH CH2 I I I CH2 I CHNH3 + CH2 COO- + AMP 2- + PPi 3- + H + I +1 t + I ATP4- l CH2 CH2 CH2 I I CH2 I COO - CH2 I I CHNH3+ CHNH3 + I I COO- CO0 - L-Citrulline L-Aspartate L-Argininosuccinate

(Chapter 27). Normally, the mitochondrial membrane is The reaction is driven forward by hydrolysis of pyrophos- not permeable to carbamoyl phosphate, but when the phate to inorganic phosphate. Argininosuccinate forma- concentration increases, carbamoyl phosphate spills into tion is considered as the rate-limiting step for urea synthe- the cytosol and promotes synthesis of orotic acid and sis. This reaction incorporates the second nitrogen atom uridine Y-phosphate. of the urea molecule donated by aspartate.

Formation of CitruUine Formation of Arginine and Fumarate Ornithine carbamoyltransferase (ornithine transcar- Argininosuccinate lyase in cytosol catalyzes cleavage bamoylase) catalyzes the condensation between car- of argininosuccinate to arginine and fumarate" bamoyl phosphate and ornithine to yield citrulline in O mitochondria: II NH z CO- NH2 NH2 / Transferred I I I I carbamoyl C,.~NH +--CH C=NH2 + NH3* C=O group I I I I I NH CH2 NH COO - CH2 NH I I I I CH 2 COO- ~ CH2 + CH I I O CH 2 CH 2 I I II II I I CH 2 CH2 HC H2N--C--O--PO3 2- + CHz ~ CH 2 + Pi 2- + H + I I I CH 2 CH2 CO0 - I I I I Carbamoyl CHNH3 + CH 2 CHNH3 + CHNH3 + phosphate I I I I COO- CHNH3 + COO- CO0- I COO - L-Argininosuccinic acid L-Arginine Fumarate L-Ornithine L-Citrulline This is the pathway for synthesis of arginine, a nonessen- Although the equilibrium constant strongly favors cit- tial amino acid; however, in the event of physiological rulline formation, the reaction is reversible. Citrulline deficiency, as in premature infants, or a defect in any of SECTION 17.2 Metabolism of Ammonia 343 the enzymes discussed above, an exogenous supply of Hyperammonemias arginine is required. Hyperammonemias are caused by inborn errors of ureagenesis and organic acidemias, liver immaturity (tran- Formation of Urea and Ornithine sient hyperammonemia of the newborn), and liver failure (hepatic encephalopathy). Neonatal hyperammonemias This irreversible reaction is catalyzed by arginase in the are characterized by vomiting, lethargy, lack of appetite, cytosol: seizures, and coma. The underlying defects can be identified by appropriate laboratory measurements (e.g., as- NH2 I sessment of metabolic acidosis if present and character- ~NH2 + NH3+ ization of organic acids, urea cycle intermediates, and I I glycine). NH CH2 I NH2 I Inborn errors of the six enzymes of ureagenesis and CH2 I CH2 NAG synthase have been described. The inheritance pat- I + H20 , C=O + I tern of the last is not known, but five of the urea cycle CH2 I CH2 defects are autosomal recessive and ornithine carbamoyl- I NH2 I CH2 CHNH3 + transferase (OCT) deficiency is X-linked. I I Carriers of OCT deficiency (estimated to be several CHNH3 § COO- thousand women in the U.S.A.) can be identified by admin- I istration of a single oral dose of allopurinol, a purine ana- CO0 - logue, followed by measurement of urinary orotidine ex- L-Arginine Urea L-Ornithine cretion. The underlying principle of this assay is that when the intramitochondrial carbamoyl phosphate accumulates The urea so formed is distributed throughout the body wa- in OCT heterozygotes, it diffuses into the cytoplasm stimu- ter and excreted. The renal clearance of urea is less than lating the biosynthesis of pyrimidines. One of the interme- the glomerular filtration rate because of passive tubular diates in this pathway--orotidine--accumulates, leading back-diffusion. Diffusion of urea in the intestine leads to to orotidinuria (Figure 17-8). formation of ammonia, which enters the portal blood and The sensitivity of this test is increased by increas- is converted to urea in liver. Reentry of ornithine into mi- ing the flux in the pyrimidine biosynthetic pathway. The tochondria initiates the next revolution of the urea cycle. enhanced flux is accomplished by allopurinol, which by Ornithine can be converted to glutamate-v-semialdehyde way of oxypurinol ribonucleotide inhibits the forma- (which is in equilibrium with its cyclic form A'-pyrroline- tion of final product uridine 5'-phosphate (UMP) in the 5-carboxylate) by ornithine aminotransferase and de- pyrimidine biosynthesis (Chapter 27). carboxylated to putrescine by ornithine decarboxylase. Antenatal diagnosis for fetuses at risk for the urea Ornithine is also produced in the arginine-glycine trans- cycle enzyme disorders can be made by appropri- amidinase reaction. ate enzyme assays and DNA analysis in the cultured The availability of substrates (ammonia and amino amniocytes. acids) in the liver determines the amount of urea syn- Acute neonatal hyperammonemia, irrespective of thesized. Urea excretion increases with increased protein cause, is a medical emergency and requires immediate intake and decreases with decreased protein intake. and rapid lowering of ammonia levels to prevent serious effects on the brain. Useful measures include hemodialy- Energetics of Ureagenesis sis, exchange transfusion, peritoneal dialysis, and admin- The overall reaction of ureagenesis is istration of arginine hydrochloride. The general goals of management are to NH3 + HCO~- + aspartate + 3ATP -+ urea + fumarate + 2ADP + 4Pi § AMP 1. Decrease nitrogen intake so as to minimize the requirement for nitrogen disposal, Hydrolysis of four high-energy phosphate groups is 2. Supplement arginine intake, and required for the formation of one molecule of urea. If 3. Promote nitrogen excretion in forms other than urea. fumarate is converted to aspartate (by way of malate and oxaloacetate), one NADH molecule is generated that can The first can be accomplished by restriction of dietary give rise to three ATP molecules through the electron protein and administration of o~-keto analogues of essen- transport chain, so that the energy expenditure becomes tial amino acids. Arginine supplementation as a precursor one ATP molecule per each molecule of urea. of ornithine is essential to the urea cycle. The diversion 344 CHAPTER 17 Protein and Amino Acid Metabolism

.('~ N-Acetylglutamate ~'~ v

[ NH 4 "~ (~ [,.' / 2,~2ATP ~ CPSI~ Carbamoyl Phosphate OTC~ I ~ Citrulline~ / ."

! Urea -" I Asparate

| i | ' Pi H2O Glutamine 1 CPS II ~r AT ~.~ gihydro- HCO 3 i> ~ CarbamoylPhosphate f v CarbamoylAspartate D "~ > Orotate 2ATP J Aspartate ) NAD

D~H.~ NADH + H §

Orotate PRPP OPRT ~ PPi Orotidine 5"-Monophosphate (OMP) .... -~ Orotidine XO PRT Oxipurinol ~ OMP AIIopurinol ~ OxipurinOlpRppf ~p~ Ribonucleotide Decarboxylase

Uridine 5"-Monophosphate

FIGURE 17-8 The metabolic interrelationship between mitochondrial carbamoyl phosphate synthesis to urea formation and to cytosolic carbamoyl phosphate channeled into pyrimidine biosynthesis. In ornithine transcarbamoylase (OTC) deficiency, mitochondrial carbamoyl phosphate diffuses into the cytosol and stimulates pyrimidine biosynthesis, leading to orotidinuria. Administration of ailopurinol augments orotidinuria by increasing the flux in the pyrimidine biosynthetic pathway. CPS = Carbamoyl phosphate synthase, AT = aspartate transcarbamoylase, D = dihydroorotase, DH -- dihydroorotate dehydrogenase, OPRT = orotate phosphoribosyltransferase, XO = xanthine oxidase, PRT = phosphoribosyltransferase, PRPP = 5-phosphoribosyl-l-pyrophosphate.

of nitrogen to products other than urea is achieved by ad- phosphate, catalyzed by mitochondrial glycine synthase ministration of sodium benzoate or sodium (or calcium) (glycine cleavage enzyme). phenylacetate. Administration of benzoate leads to elimi- Phenylacetate or phenylbutyrate administration in- nation of hippurate (benzoylglycine): creases excretion of phenylacetylglutamine:

O O )--CH3--COO- + ATP4- + CoASH Activating enzyme CH3MC~SCoA + AMPz - + PPi3- ~~'-~nO- + ATP 4- + CoASH Acfivalingenzyme PhenylacetyI-CoA

Benzoate CONH2 CONH2 l I 0 (CH~)~ O O (0H2)2 Conjugatmgenzyme,/g~=.~CH3MC__NBCH II I + CoASH+ H + II I ' H I CH3BC~SCoA + H3+N- CH ~~--~mSmCoA + AMp2- + PPi 3- I COO - COO - BenzoyI-CoA Glutamine Phenylacetylglutamine The excretion of phenylacetylglutamine produces loss of Hippurate is rapidly secreted since its clearance is five two nitrogen atoms. times greater than its glomerular filtration rate. The glycine NAG synthase deficiency cannot be treated by admin- nitrogen is derived from ammonia in a complex reaction istration of NAG, since NAG undergoes cytosolic inacti- that uses CO2, NADH, NS,Nl~ vation by deacylation and is not readily permeable across (a source of a single carbon unit; Chapter 27) and pyridoxal the inner mitochondrial membrane. An analogue of NAG, SECTION 17.3 Metabolism of Some Individual Amino Acids 345

N-carbamoylglutamate, activates CPSI, does not share the mediate in the urea cycle pathway and is also obtained undesirable properties of NAG, and has been effective in from dietary proteins. A number of key metabolites such management of this deficiency. as nitric oxide, phosphocreatine, spermine and ornithine The most common cause of hyperammonemia in adults are derived from arginine. During normal growth and de- is disease of the liver (e.g., due to ethanol abuse, infection, velopment, under certain pathological conditions (e.g., en- or cancer). The ability to detoxify ammonia is decreased dothelial dysfunction) and if the endogenous production in proportion to the severity of the damage. In advanced of arginine is insufficient, a dietary supplement of arginine disease (e.g., cirrhosis), hyperammonemia is augmented may be required. Thus, arginine is considered a semiessen- by shunting of portal blood that carries ammonia from the tial amino acid. intestinal tract and other splanchnic organs to the systemic blood circulation (bypassing the liver) and leads to portal- systemic encephalopathy. In addition to dietary protein Metabolism and Synthesis of Nitric Oxide restriction, colonic growth of bacteria must be suppressed Nitric oxide (NO) is a reactive diatomic gaseous molecule by antibiotics (e.g., neomycin) and administration of lac- with an unpaired electron (a free radical). It is lipophilic tulose (Chapter 9), a nonassimilable disaccharide. Enteric and can diffuse rapidly across biological membranes. NO bacteria catabolize lactulose to organic acids that convert mediates a variety of physiological functions such as en- NH3 to NH4+, thereby decreasing absorption of NH3 into dothelial derived relaxation of vascular smooth muscle, the portal circulation. Catabolism of lactulose also leads to inhibition of platelet aggregation, neurotransmission, and formation of osmotically active particles that draw water cytotoxicity. The pathophysiology of NO is a double- into the colon, produce loose, acid stools, and permit loss edged sword. Insufficient production of NO has been im- of ammonia as ammonium ions. plicated in the development of hypertension, impotence, susceptibility to infection, and atherogenesis. Excessive NO production is linked to septic shock, inflamma- 17.3 Metabolism of Some Individual Amino Acids tory diseases, transplant rejection, stroke, and carcino- genesis. Mammalian tissues synthesize the nonessential amino NO is synthesized from one of the terminal nitrogen acids from carbon skeletons derived from lipid and car- atoms or the guanidino group of arginine with the con- bohydrate sources or from transformations that involve comitant production of citrulline. Molecular oxygen and essential amino acids. The nitrogen is obtained from NADPH are cosubstrates and the reaction is catalyzed NH4+ or from that of other amino acids. Nonessential by nitric oxide synthase (NOS). NOS consists of sev- amino acids (and their precursors) are glutamic acid eral isoforms and is a complex enzyme containing bound (ot-ketoglutaric acid), aspartic acid (oxaloacetic acid), FMN, FAD, tetrahydrobiopterin, heme complex, and non- serine (3-phosphoglyceric acid), glycine (serine), tyro- heme iron. A calmodulin binding site is also present. NO sine (phenylalanine), proline (glutamic acid), alanine formation from arginine is a two-step process requiring (pyruvic acid), cysteine (methionine and serine), arginine five-electron oxidations. The first step is the formation of (glutamate-y-semialdehyde), glutamine (glutamic acid), NC-hydroxylarginine (N G denotes guanidinium nitrogen and asparagine (aspartic acid). atom): Amino acids may be classified as ketogenic, glucogenic, or glucogenic and ketogenic, depending on whether feed- Arginine + O2 + NADPH + H + -+ ing of a single amino acid to starved animals or animals HO-NC-Arg + NADP + + H20 with experimentally induced diabetes increases plasma or urine levels of glucose or ketone bodies (Chapter 18). This step is a mixed-function oxidation reaction simi- Leucine and lysine are ketogenic; isoleucine, phenylala- lar to the one catalyzed by cytochrome P-450 reduc- nine, tyrosine, and tryptophan are glucogenic and keto- tase and there is considerable homology between NOS genic; and the remaining amino acids are glucogenic. and cytochrome P-450 reductase. In the second step, fur- Points of entry of amino acids into the gluconeogenic path- ther oxidation of NG-hydroxyl arginine yields NO and way are discussed in Chapter 15. citrulline: 1 Arginine HO-NG-Arg + 02 -~- ~(NADPH + H +) --+ 1 Arginine participates in a number of metabolic pathways citrulline + NO + H20 -k- -NADP + depending on the cell type. It is synthesized as an inter- 2 346 CHAPTER 17 Protein and Amino Acid Metabolism

The overall reaction is" interferon-v), or bacterial lipopolysaccharides can induce O H ?H 2 and cause expression of NOS in many cell types. Glu- II I "Om C n OH m (0H2)3~ N -- C--NH + 0 2 + NADPH + H § cocorticoids inhibit the induction of iNOS. In stimulated I macrophages and neurophils, NO and superoxide radi- NH3+ cal (O2) react to generate peroxynitrite, a powerful ox- Arginine idant, and hydroxyl radicals. These reactive intermedi- Nitric Oxide / FMN, FAD, Tetrahydrobiopterin ates are involved in the killing of phagocytized bacteria Synthase 1 Fe 2+, Heme complex (Chapter 14). Excessive production of NO due to endotox- inemia produces hypotension and vascular hyporeactivity to vasoconstrictor agents, and leads to septic shock. NOS o H O II I II inhibitors have potential therapeutic application in the -O-- C -- OH -- (OH2) 3- N -- C -- NH 2 + NO treatment of hypotensive crisis. I NH3+ Citrulline Signal Transduction of NO The NOS activity is inhibited by NO-substituted analogues of arginine, such as NC-nitroarginine and N c- NO is lipophilic and diffuses readily across cell mem- monomethyl-L-arginine. branes. It interacts with molecules in the target cells pro- ducing various biological effects. One mechanism of action of NO is stimulation of guanylate cyclase, which Isoforms (Also Known as Isozymes) catalyzes the formation of cyclic guanosine monophos- of Nitric Oxide Synthase phate (cGMP) from GTP, resulting in increased intracel- There are three major isoforms of Nitric Oxide lular cGMP levels (Figure 17-9). NO activates guanylate Synthase (NOS) ranging in molecular size from 130 to cyclase by binding to heme iron. The elevation of cGMP 160 kDa. Amino acid similarity between any two isoforms levels may activate cGMP-dependent protein kinases. is about 50-60%. Isoforms of NOS exhibit differences in tissue distribution, transcriptional regulation, and activa- O tion by intracellular Ca 2+. Two of the three isoforms of NOS are constitutive enzymes (cNOS) and the third iso- II II II forin is an inducible enzyme (iNOS). The cNOS isoforms HN "~0\5,2~ O-- I~--0-- P--O--I.P --O- H"~H H//~,H O- O- O- are found in the vascular endothelium (eNOS), neuronal F--V-' OH OH cells (nNOS), and many other cells, and are regulated Derived from by Ca 2+ and calmodulin. In the vascular endothelium, Guanosine Triphosphate (GTP) Nerve Impulse 7 / agonists such as acetylcholine and bradykinin activate J eNOS by enhancing intracellular Ca 2+ concentrations PPi j GuanylateCyclase ~ ~ NO via the production of inositol 1,4,5-trisphosphate, which O Derived from a activates the phosphoinositide second-messenger system Ligand-cell interaction HN (Chapter 30 ). The NO produced in the vascular endothelium maintains basal vascular tone by vasodilation which H2N : o is mediated by vascular smooth muscle cells. Organic .,> Smooth muscle .. relaxation O nitrates used in the management of ischemic heart disease , /CH 3 act by denitration with the subsequent formation of NO. Cyclic GMP CH3CH20 HN~N\ .... I /; Sodium nitroprusside, an antihypertensive drug, is an NO Cyclic GMP donor. Thus, organic nitrates and sodium nitroprusside phosphodiesterase ~ N~CH2CH2CH3 are prodrugs, and the exact mechanism by which these O O2S\ N "~ prodrugs yield NO is not yet understood. Inhaled NO can HN N%, O ~. produce pulmonary vasodilation. This property of NO CH3 H2N - ~'N / -Nk/o 5" C -- O-- P-- O- Sildenafil has been used in the management of hypoxic respiratory H,,/ H~H O-I failure associated with primary pulmonary hypertension OH OH in neonates. NO produced by cNOS in neuronal tissue 5"-GMP functions as a neurotransmitter. The inducible class of NOS (iNOS) is found in FIGURE 17-9 NO-mediated synthesis of cGMP from GTP in the corpus cavernosum that macrophages and neutrophils and is Ca2+-independent. leads to smooth muscle relaxation. Sildenafil potentiates the effects of NO Bacterial endotoxins, cytokines (e.g., interleukin-1, by inhibiting cGMP phosphodiesterase. SECTION17.3 Metabolism of Some Individual Amino Acids 347

Purines --Glucose Proteins\\ 3-PhosphoglyceratJ/ Urea_ Glutathione~ \\ f ~ Pyruvate "One-carbon""~~" + NH~'+ "~\~~GlyciEe ,_ | ~ ~TCA J/~"/~~ Seri ne~ Ethan~amine.,~ cycle-----* C O2 Bile acid conjugates oI line

other acylHippurate conjugatesand /~/ ~ Glyoxalatel . Glycolate" " Phospholipids Porphyrins Creatine Oxalate Phosphocreatine1 1 Creatinine FIGURE 17-11) Overview of glycine and serine metabolism.

These kinases phosphorylate specific proteins that may The antiaggregability of platelets and the neurotoxicity of be involved in removal or sequestration of Ca 2+ or other NO have been attributed to inhibition of glycolysis by NO. ions, resulting in physiological stimuli. The physiological actions of cGMP are terminated by its conversion to Glycine 5'-GMP by cGMP-phosphodiesterase. Inhibitors of cGMP participates in a number of synthetic path- phosphodiesterase promote the actions of NO. Glycine ways and is oxidized to provide energy (Figure 17-10). Sildenafil is a selective inhibitor of a specific cGMP The interconversion of glycine and serine by serine phosphodiesterase (type 5) present in the corpus caver- hydroxymethyltransferase is shown below: nosum. This compound (structure shown in Figure 17-9) is used orally in the therapy of some types of erectile dys- pyridoxal phosphate NH~--CH2-COO- + N 5 ,N 10 -methylene-FH4 + H20 < > function. NO is the principal transmitter involved in the glycine relaxation of penile smooth muscle. During central or re- HOHzC-CHNH+-COO - + FH4 flex sexual arousal, NO production is enhanced leading serine to increased production of cGME Smooth muscle relax- The one-carbon carrier NS,Nl~ ation permits the corpus cavernosum to fill with blood. is derived from reactions of the one-carbon pool (Chap- Since the therapeutic effect of sildenafil potentiates the ter 27). [The term one-carbon pool refers to all action of cGMP, the drug is ineffective in the absence of single-carbon-containing metabolites (e.g.,-CH3,-CHO, sexual arousal. The relaxation of cavernosal smooth mus- NH=C-, etc.) that can be utilized in biosynthetic reac- cle caused by cGMP involves inhibition of Ca 2+ uptake. tions such as formation of purine and pyrimidine.] These Prostaglandin E1 (alprostadil) inhibits the uptake of Ca 2+ reactions include oxidation of glycine by glycine cleavage smooth muscle by a separate mechanism and causes erec- enzyme complex (glycine synthase): tions in the absence of sexual arousal. Blood flow through corpus cavernosum may also be increased by o~-adrenergic pyridoxal phosphate blocking agents (e.g., phentolamine mesylate). Coadmin- NH+-CH2-COO - nt- FH4 + NAD + < > istration of NO donor drugs with the NO potentiation drug glycine sildenafil may have severe consequences on the cardiovas- NH + + C02 + NADH + NS,Nl~ cular system. Signal transduction of NO by cGMP-independent This reaction favors glycine degradation, but the formation mechanisms include ADP-ribosylation of glyceraldehyde- of glycine may also occur. The enzyme complex is mito- 3-phosphate dehydrogenase (GADPH), an enzyme of the chondrial and contains a pyridoxal phosphate-dependent glycolytic pathway (Chapter 13), and interactions with glycine decarboxylase, a lipoic acid-containing pro- many heme-containing and nonheme iron-sulfur contain- tein that is a carrier of an aminomethyl moiety, a ing proteins. NO activates ADP-ribosyltransferase which tetrahydrofolate-requiring enzyme, and lipoamide catalyzes the transfer of ADP-ribose from NAD + to dehydrogenase. The reactions of glycine cleavage re- GADPH. This results in the inactivation of GADPH caus- semble those of oxidative decarboxylation of pyruvate ing inhibition of glycolysis and decreased ATP production. (Chapter 13). 348 CHAPTER 17 Protein and Amino Acid Metabolism

Glycine is also oxidized by D-amino acid oxidase, an Creatine and Related Compounds FAD protein: Phosphocreatine serves as a high-energy phosphate donor for ATP formation (e.g., in muscle contraction; see NH~--CH2-COO- -Jr- 02 -~- H20 glycine Chapter 21). Synthesis of creatine (methyl guanidinoacetate) requires transamidination, i.e., transfer of a guani- CHO-COO- + NH + + H202 glyoxalate dine group from arginine to glycine, to form guanidinoacetate (glycocyamine) by mitochondrial arginine-glycine Glyoxalate can be transaminated to glycine, reduced to amidinotransferase glycolate, converted to ot-hydroxy-/~-ketoadipate by reac- NH2 tion with o~-ketoglutarate, or oxidized to oxalate and ex- I NH2 creted in urine. The first three reactions require pyridoxal ~NH2 + I NH3 + I NH3 + ~NH2+ I phosphate, NADH, and thiamine pyrophosphate, respec- NH I I (CH2)3 tively. In humans, ascorbic acid (vitamin C) is a precursor I + CH 2 ~ '~ NH I of urinary oxalate (Chapter 38). Since calcium oxalate is (CH2)3 I I CHNH3 I COO- CH2 I poorly soluble in water, it can cause nephrolithiasis and CHNH3 + I CO0 - nephrocalcinosis due to hyperoxaluria. I coo- COO -

Arginine Glycine Guanidinoacetate Ornithine Disorders of Glycine Catabolism (glycocyamine) Nonketotic hyperglycinemia is an inborn error due to a defect in the glycine cleavage enzyme complex in which In the next step guanidinoacetate is methylated by S- glycine accumulates in body fluids and especially in cere- adenosylmethionine by cytosolic S-adenosylmethionine brospinal fluid. It is characterized by mental retardation guanidinoacetate-N-methyltransferase to form creatine. and seizures. Glycine is an inhibitory neurotransmitter OH 3 NH2 NH2 in the central nervous system, including the spinal cord. I I I S-Adenosyl Strychnine, which produces convulsions by competitive S+.Adenosyl C--NH2 + C--NH2 + I I [ I (CH2)2 inhibition of glycine binding to its receptors, gives mod- (0H2)2 + NH ~ N--OH3 + I + H + I I I CHNH3 § est results in treatment but not very effective. Sodium CHNH3 + CH2. CH2 I benzoate administration reduces plasma glycine levels but I I I coo - COO- COO- COO- does not appreciably alter the course of the disease. Ex- change transfusion may be useful. Ketotic hyperglycine- S-Adenosyl- Guanidino- Creatine S-Adenosyl- methionine acetate homocysteine mia also occurs in propionic acidemia but the mechanism has not been established. These reactions occur in liver, kidney, and pancreas, from Primary hyperoxaluria type I is due to a deficiency of which creatine is transported to organs such as muscle cytosolic oe-ketoglutarate-glyoxylate carboligase, which and brain. Creatine synthesis is subject to negative modu- catalyzes the following reaction: lation of amidinotransferase by creatine. Phosphocreatine production is catalyzed by creatine kinase: O II NH2 NH--PO3 2- CHOmCOO- + - OOC__CH2~CH2wC..._COO - I I C--NH2 + C=NH2 + Glyoxalate 0c-Ketoglutarate I I N--CH 3 + ATP 4- ~ N--CH 3 + ADP 3- + H + I I CH2 CH 2 Carboligase Thiaminepyrophosphate I I COO- COO-

Creatine Phosphocreatine O OH II I This kinase is a dimer of M and B (M = muscle, B = CO2 + -OOC~CH2mCH2--C~CH~COO- brain) subunits produced by different structural genes. 0e-Hydroxy-/Y-ketoadipate Three isozymes are possible: BB (CK-1), MB (CK-2), and MM (CK-3). Another isozyme differs immunologi- The glyoxylate that accumulates is converted to cally and electrophoretically and is located in the inter- oxalate. membrane space of mitochondria. Tissues rich in CK-1 SECTION 17.3 Metabolism of Some Individual Amino Acids 349 are brain, prostate, gut, lung, bladder, uterus, placenta, regenerates ATP from ADP, thereby maintaining a high and thyroid; those rich in CK-3 are skeletal and car- level of ATP required during intense exercise. A large pool diac muscle. Cardiac muscle contains significant amounts of phosphocreatine resides in the skeletal muscle. It has of CK-2 (25-46% of total CK activity, as opposed to been theorized that in order to maximize phosphocreatine less than 5% in skeletal muscle), so that in myocardial stores in the skeletal muscle to replenish ATP during rapid infarction the rise in serum total CK activity is accompa- muscle contractions, an exogenous source of creatine may nied by a parallel rise in that of CK-2 (Chapter 8). be beneficial. Phosphocreatine undergoes a slow and nonenzymatic Double-blind placebo-controlled studies of oral sup- cyclization to creatinine. plementation of creatine in human subjects have shown increased performance during short duration, strenuous, NH~PO3 2- high-intensity exercise. Such activities require that ATP I ~NH2, NH be replenished rapidly from phosphocreatine stores during I H2N---C ~~O anaerobic metabolism. These studies usually consisted of N--CH3 +H* , \ / + Pj~- + H20 ingestion of 20 g of creatine per day for 5 days followed I CH2 N~CH2 by a maintenance dose of 5-10 g/day. Studies on crea- j / tine as an ergogenic aid have not been uniformly positive; COO- HaC some have shown no beneficial effect and still others have Phosphocreatine Creatinine been equivocal and indicated that creatine supplementation did not enhance athletic activities. The safety issues Creatinine has no useful function and is eliminated by of long-term creatine supplementation on kidney, liver, renal glomerular filtration and to a small extent by renal nerve, muscle, and other tissues are not known. tubular secretion. Creatinine clearance approximately par- allels the glomerularfiltration rate (GFR) and is used as Serine a kidney function test. It is calculated as follows: Synthesis of serine from 3-phosphoglycerate, an inter- urine creatinine (mg/L) mediate of glycolysis (Chapter 13), requires oxidation Creatinine clearance = plasma creatinine (mg/L) of 3-phosphoglycerate to 3-phosphohydroxypyruvate, • urine volume per unit time transamination of 3-phosphohydroxypyruvate by glutamate, and hydrolysis of 3-phosphoserine to serine Creatinine concentrations are measured from a pre- (Figure 17-11). This cytosolic pathway is regulated by cisely timed urine specimen (e.g., 4-hour, 24-hour) and inhibition of phosphoserine phosphatase by serine. Serine a plasma specimen drawn during the urine collection is converted to pyruvate by cytosolic serine dehydratase. period. Excretion of creatinine depends on skeletal mus- More importantly, it is converted in mitochondria to cle mass and varies with age and sex. However, day-to- 2-phosphoglycerate by way of hydroxypyruvate and day variation in a healthy individual is not significant. D-glycerate; and the enzymes involved are a transam- Creatinuria, the excessive excretion of creatine in urine, inase, a dehydrogenase, and a kinase. Serine is in- may occur during growth, fever, starvation, diabetes melli- terconvertible with glycine (Figure 17-10) and is in- tus, extensive tissue destruction, muscular dystrophy, and volved in phospholipid (Chapter 19) and in cysteine hyperthyroidism. synthesis.

Use of Creatine as a Dietary Supplement Proline The creatine pool in the human body comes from both Proline arises from and gives rise to glutamate. Synthesis endogenous synthesis and the diet which provides 1-2 g. is by reduction of glutamate to glutamate-v-semialdehyde Red meat provides large amounts of dietary creatine and by way of an enzyme-bound v-glutamyl phosphate. The vegetables a limited amount. Using glycine, arginine, and v-semialdehyde spontaneously cyclizes to A'-pyrroline- methionine, creatine is synthesized in the liver, pancreas, 5-carboxylate, which is then reduced by NAD(P)H to pro- and kidney. Creatine transported in blood crosses muscle line (Figure 17-12). Proline is converted to A'-pyrroline-5- and nerve cell membranes by means of a specific crea- carboxylate by proline oxidase, which is tightly bound to tine transporter system against a concentration gradient of the inner mitochondrial membrane in liver, kidney, heart, 200:1. Intracellularly, creatine is converted to phosphocre- and brain. A'-Pyrroline-5-carboxylate is in equilibrium atine by ATE a reaction catalyzed by creatine kinase. Phos- with glutamate-v-semialdehyde, which can be transami- phocreatine, with its high phosphoryl transfer potential, nated to ornithine or reduced to glutamate (Figure 17-12). 350 CHAPTER17 Protein and Amino Acid Metabolism

coo- coo- l Phosphoglycerate I Glycolysis-"-"-"-~ HC-- OH dehydrogenase , H --O--(~ NAD+ NADH+ H2C--O--(~ 3-Phosphoglycerate 3-Phosphohydroxypyruvate t~nG lutamate osphoserine saminase ec-Ketoglutarate COO- Phosphoserine COO- , H3N?HI -_ phosphatase(,,.~-'~ H2N~H CH~----OH P~ H O ~C--O--(~ L-Serine 3-Phosphoserine FIGURE 17-11 Synthesis of serine from 3-phosphoglycerate. P = PO 2-" Pi -- HPO 2-.

Decarboxylation of ornithine to putrescine by ornithine decarboxylase. Ornithine aminotransferase deficiency is decarboxylase serves as a source of the polyamines associated with gyrate atrophy of the choroid and retina spermidine and spermine. Ornithinemia results from (Chapter 38). Proline and hydroxyproline (produced by deficiency of ornithine aminotransferase or ornithine posttranslational modification) are major constituents

CO0- 1 ATP (? H2)= ADP,~...... ------~ ? HNH+ COO- NADPH+ H+.~/(~" L-Glutamate NAOP ) CHO ._. / I (~/--- § (C,H,)~ ~ NADH+ H ICHNH+ ~ NAD+ ICOO- ~~H20 I-I=? ?Hz Glutamate-7-semialdehyde _ l,,-----Glutamate H20 HC~N/CHCOOH ('~F er -Ketoglutarate A -Pyrroline- ?(rH2NH+ H+~ 5"carbOxylate

(H~ C02 NAD(P)H+ CHNH+ 3 , ?HF---NH+ Flav%iotein I | NAD(P)*-~ COO- (?Hz), Ornithine CH=NH+ Via ~ C CH2 reactionsof Putrescine H2C~N/CHCOOHI urea cycle Arginine H Proline FIGURE 17-12 Metabolism of proline (1) A1-pyrroline-5-carboxylate (P5C) synthase; (2) ornithine aminotransferase; (3) P5C reductase; (4) proline oxidase; (5) P5C dehydrogenase; (6) ornithine decarboxylase. SECTION 17.3 Metabolism of Some Individual Amino Acids 351 of collagen (Chapter 25). Hydroxyproline released by NS-methyltetrahydrofolate and is unavailable as a carrier collagen turnover undergoes degradation similar to for the formimino group of Figlu. that of proline. Hydroxyproline cleavage initiated by hydroxyproline oxidase eventually yields glyoxylate and Homocysteine methyltransferase NS-Methy I-FH 4 (methylcobalamin) pyruvate. In hyperprolinemia type I, proline oxidase /- ~ ~ FH4 is deficient, and in type II, A'-pyrroline-5-carboxylate Homocysteine Methionine dehydrogenase is deficient. Hydroxyprolinemia results Similarly, a deficiency of glutamate formiminotransferase from hydroxyproline oxidase deficiency. All are clinically leads to accumulation of Figlu and high levels of serum harmless autosomal recessive traits. folate. Histidinemia results from deficiency of histidine Histidine ammonia-lyase. With a normal diet, histidine (and the pro- Histidine is not essential for adults except in ducts imidazole-pyruvate, imidazole-lactate, imidazole- persons with uremia. It is essential for growth in acetate) accumulates in plasma, cerebrospinal fluid, and children. Histidine is synthesized from 5-phosphoribosyl- urine. This rare autosomal recessive disease may be 1-pyrophosphate and ATE forming N'-l'-phosphoribosyl- benign or may manifest with mental retardation and speech ATE catalyzed by the allosteric enzyme ATP phosphori- defects. bosyltransferase. This reaction is analogous to the initial Histidine and #-alanine yield the dipeptide carnosine reaction of purine nucleotide biosynthesis (Chapter 27). (present in muscle), and histidine and 7-aminobutyrate Histamine breakdown produces a one-carbon unit (N 5- yield homocarnosine (found in brain). Methylhistidyl resi- formiminotetrahydrofolate) and glutamate (Figure 17-13) dues are found in some proteins (e.g., actin; Chapter 21) by a nonoxidative deamination to urocanate, cleavage of as a result of posttranslational modification. Histamine is the imidazole ring to N-formiminoglutamate (Figlu), and decarboxylated histidine. transfer of the formimino group (-CH=NH) to tetrahydrofolate (Chapter 27). H Histidine decarboxylase I (pyridoxal phosphate) Folate deficiency leads to accumulation of Figlu, which )[ ] CH2--CH2--NH3+ is excreted in urine. The excretion is very pronounced N~.~,,,,, N H COO- N~,,~,,,/N H after a loading dose of histidine, a test used to detect fo- CO2 late deficiency. More sensitive radioisotopic assays use fo- Histidine Histamine late binders to the vitamins. High urinary levels of Figlu Histamine occurs in blood basophils, tissue mast cells, may coexist with elevated levels of serum folate. Thus, in and certain cells of the gastric mucosa and other parts of vitamin B]2 (cobalamin) deficiency, since cobalamin the body (e.g., anterior and posterior lobes of the pituitary, participates in the following reaction, FH4 is trapped as some areas of the brain). Histamine is a neurotransmitter in certain nerves ("histaminergic") in the brain. In mast cells found in loose connective tissue and capsules, especially NH + around blood vessels, and in basophils, histamine is stored I Histidine------~CHF-CH--COO- ammonia-lyase ,. in granules bound by ionic interactions to a heparin-protein (histidase) I]--C"=CHCOO N~.~/NH NH3 N~,,.,/.NH complex and is released (by degranulation, vacuoliza- L-Histidine Urocanate tion, and depletion) in immediate hypersensitivity reac- H20-~ Urocanate O H hydratase tions, trauma, and nonspecific injuries (infection, burns). II I Imidazolone- 0~] Degranulation is affected by oxygen, temperature, and -O--C--C--CH~--C~COO- =~oropionase ~1 CHF"CH2COO- I metabolic inhibitors. Release of histamine from gastric HN ~c./NH ~0 N~NH I mucosal cells is mediated by acetylcholine (released by H 4-1midazolone-5-propionate parasympathetic nerve stimulation) and gastrin and stim- N-Formiminoglutamate (Figlu) Glutamate ~FH, ulates secretion of hydrochloric acid (Chapter 12). His- formiminotransferase NLformino.FH' tamine causes contraction of smooth muscle in various O organs (gut, bronchi) by binding to H1 receptors. The II -O-- C-- CH--CH.~H2COO - conventional antihistaminic drugs (e.g., diphenhydramine and pyrilamine) are Hi-receptor antagonists and are use- L-Glutamate ful in the management of various allergic manifestations. FIGURE 17-13 However, in acute anaphylaxis, bronchiolar constriction is Catabolism of histidine. rapidly relieved by epinephrine (a physiological antagonist 352 CHAPTER 17 Protein and Amino Acid Metabolism of histamine). Its effect on secretion of hydrochloric acid is mediated by H2 receptors. Hz-receptor antagonists are cimetidine and ranitidine (Chapter 12), which are useful in treatment of gastric ulcers. Histamine is rapidly inactivated by methylation from S-adenosylmethionine of one of the nitrogen atoms of the imidazole ring (catalyzed by N-methyltransferase) or of the terminal amine group (catalyzed by methyltransferase). Ring-methylated histamine is deaminated by monoamine oxidase to methyl imidazole acetic acid, which is readily excreted. Inactivation also results from deamination of histamine by diamine oxidase. The imidazole acetic acid formed is then excreted as 1- ribosylimidazole-4-acetic acid. This reaction is the only known reaction in which ribose is used for conjugation.

Branched-Chain Amino Acids Leucine, isoleucine, and valine are essential amino acids but can be derived from their respective ot-keto acids. A single enzyme may catalyze transamination of all three. FIGURE 17-14 The ot-keto acids, by oxidative decarboxylation, yield Overview of the catabolism of branched-chain amino acids. TPP = thiamin the acyl-CoA thioesters, which, by ot,/3-dehydrogenation, pyrophosphate. yield the corresponding ot,fl-unsaturated acyl-CoA thioesters. The catabolism of these thioesters then di- verges. Catabolism of leucine yields acetoacetate and Hypoglycin produces hypoglycemia and metabolic aci- acetyl-CoA via fl-hydroxy-fl-methylglutaryl-coenzyme dosis, which frequently are fatal. A (HMG-CoA)malso an intermediate in the biosynthe- Branched-chain ketoaciduria (maple syrup urine dis- sis of cholesterol and other isoprenoids (Chapter 19). ease), an autosomal recessive disorder characterized by Catabolism of isoleucine yields propionyl-CoA (a gluco- ketoacidosis starts early in infancy and is due to a de- genic precursor) and acetyl-CoA. Catabolism of valine fect in the oxidative decarboxylation step of branched- yields succinyl-CoA (Figure 17-14). Thus, leucine is chain amino acid metabolism. The name derives from ketogenic and isoleucine and valine are ketogenic and the characteristic odor (reminiscent of maple syrup) of glucogenic. the urine of these patients. Five different variants (clas- Oxidative decarboxylation of the c~-keto acids is cat- sic, intermittent, intermediate, thiamine-responsive, and alyzed by a branched-chain keto acid dehydrogenase dihydrolipoyl dehydrogenase deficiency) are known, of (BCKADH) complex analogous to that of the pyru- which the first, which is due to deficiency of branched- vate dehydrogenase and c~-ketoglutarate dehydrogenases chain ot-keto acid decarboxylase, is the most severe. The complexes. BCKADH is widely distributed in mam- incidence of maple syrup urine disease in the U.S. popu- malian tissue mitochondria (especially in liver and kid- lation is 1 in 250,000-400,000 live births. In Mennonite ney). It requires Mg 2+, thiamine pyrophosphate, CoA-SH, populations the incidence is extremely high (1 in 760). lipoamide, FAD, and NAD + and contains activities of ot- Neonatal screening programs consist of measuring leucine keto acid decarboxylase, dihydrolipoyl transacylase, and levels in dried blood spots using a bacterial inhibition as- dihydrolipoyl dehydrogenase. Like the pyruvate dehydro- say. Neonatal screening programs usually include testing genase complex, BCKADH is regulated by product in- for a number of other treatable metabolic disorders such hibition and by phosphorylation (which inactivates) and as hypothyroidism, phenylketonuria, galactosemia, and dephosphorylation (which activates). others. If the screening test is positive for a given metabolic The o~,fl-dehydrogenation is catalyzed by an FAD pro- disease, a confirmatory test is performed. For maple syrup tein and is analogous to the dehydrogenation of straight- urine disease, the confirmation requires quantitation of chain acyl-CoA thioesters in fl-oxidation of fatty acids the serum levels of branched-chain amino acids and urine (Chapter 18). Methylenecyclopropylacetyl-CoA derived levels of both the branched-chain amino acids and their from the plant toxin hypoglycin (Chapters 15 and 18), ketoacids. Long-term management includes dietary re- which inhibits this step in fl-oxidation, also inhibits striction of the branched-chain amino acids. Frequent it in the catabolism of branched-chain amino acids. measurement of plasma concentrations of these amino SECTION 17.3 Metabolism of Some Individual Amino Acids 353 acids is necessary to monitor the degree of dietary restric- NH2 NH2 + I I tion and patient compliance. Adenosine ~ S~ (CH=)z~CH~ COOH Adenosine ~ S~ (CH2);--CHmCOOH / Many aminoacidurias and their metabolites give rise CH3 to abnormal odors, maple syrup urine disease is one ex- S-Adenosylmethionine S-Adenosylhomocysteine . Specificmethyltransferase ample. Some of the others are phenylketonuria (musty odor), tyrosinemia type I (boiled cabbage), glutaric Acceptor (examples) Acceptor ~ CH, (examples) aciduria (sweaty feet), 3-methylcrotonylglycinuria (cat's 1. Guanidinoacetic acid 1. Creatine 2. Nicotinamide 2. N-Methylnicotinamide urine), and trimethylaminuria (fish). In patients with 3. Norepinephrine 3. Epinephrine 4. Phosphatidylethanolamine 4. Phosphatidylcholine trimethylaminuria the compound responsible for the fish 5. N-Acetyl-serotonin (three cycles of methylation) 5. Melatonin odor is trimethylamine which is a byproduct of protein FIGURE 17-15 catabolism by the large intestinal bacterial flora. Normally, Selected methyl transfer reactions involving S-adenosylmethionine. trimethylamine is inactivated by hepatic flavin monooxygenases. Several different mutations in the gene for flavin monooxygenases have been identified in trimethylamin- The methyl group is transferred to appropriate accep- uric patients. An inhibitor of flavin monooxygenases is tors by specific methyltransferases with production of indole-3-carbinol found in dark green vegetables (e.g., S-adenosylhomocysteine (Figure 17-15), which is hy- broccoli). The amelioration of symptoms of bad odor in drolyzed to homocysteine and adenosine by adenosylho- trimethylaminuria may be achieved by limiting intake of mocysteinase: dark green vegetables and protein, and by administering low doses of antibiotics to reduce intestinal bacterial flora. NH3 + NH3 + I H20 Adenosine I AdenosyI--S--(CH2)2--CHmCOO- ~ J : HS~(CH2)2~ CHmCOO-

S-Adenosylhomocysteine Homocysteine Sulfur-Containing Amino Acids Homocysteine can be recycled back to methionine Methionine and cysteine are the principal sources of or- either by transfer of a methyl group from betaine catalyzed ganic sulfur in humans. Methionine is essential (unless by betaine-homocysteine methyltransferase, or from N 5- adequate homocysteine and a source of methyl groups are methyltetrahydrofolate (NS-methyl-FH4) catalyzed by N 5- available), but cysteine is not, since it can be synthesized methyl-FH4-methyltransferase, which requires methyl from methionine. cobalamin:

(:) NH3 + ! Betaine-homocysteine Methionine HS~(CH2)2~H~COO- + (CH3)3+N- CH2~COO- methyltransferase ,

Methionine is utilized primarily in protein synthe- Homocysteine Betaine sis, providing sulfur for cysteine synthesis, and is the NH3 + body's principal methyl donor. In methylation reactions, I S-adenosylmethionine (SAM) is the methyl group donor. H3C__S__(Ch2)2mCH~COO- + (CH3)2N--CH2--COO- SAM is a sulfonium compound whose adenosyl moiety is Methionine Dimethylglycine derived from ATP as follows: (2) NH 3+ NS_Methyt_FH4.homocysteine methyltransferase I (methylcobalamin) NH3 + HS~(CH2)2~CH~COO- + NS~MethyI~FH4 I ATP.H~ ~.spp, + p. CH3~S~(CH2)2--CH~COO NH3 + Methionine adenosyltransferase I Methionine H3CmSm(CH2)2--CHmCOO - + FH4

Betaine (an acid) is obtained from oxidation of choline NH2 (an alcohol) in two steps"

NH3 + I FAD FADH2 -OOC--CH--(CH2)2-- ~; CH2 I (CH3)3+N__CH2--CH2OH ~ J ,(CH3)3+N__CH2mCHO I Choline oxidase CH3 Choline Betaine aldehyde NAD*~I '~ Belaine aldehyde .,/1 dehydr~ HO OH NADH + H+Wl S-Adenosylmethionine (CH3)3N__CH2mCOO - (active methionine) Betaine 354 CHAPTER17 Protein and Amino Acid Metabolism

Cysteine carboxylic acid group. Taurine is conjugated with bile In the biosynthesis of cysteine, the sulfur comes from acids in the liver (Chapter 19) and is readily excreted by the methionine by transsulfuration, and the carbon skeleton kidney. It is a major free amino acid of the central nervous and the amino group are provided by serine (Figure 17-16). system (where it may be an excitatory neurotransmitter) Cysteine regulates its own formation by functioning and the most abundant in the retina; it also occurs in other as an allosteric inhibitor of cystathionine v-lyase, ot- tissues (e.g., muscle, lung). Ketobutyrate is metabolized to succinyl-CoA by way of Sulfate can be converted to the sulfate donor compound propionyl-CoA and methylmalonyl-CoA. 3'-phosphoadenosine-5'-phosphosulfate (PAPS) in a two- Cysteine is required for the biosynthesis of glu- step reaction (Figure 17-17). PAPS participates in the tathione and of CoA-SH. A synthetic derivative, N- sulfate esterification of alcoholic and phenolic functional acetylcysteine, is used to replenish hepatic levels of glu- groups (e.g., in synthesis of sulfolipids and glycosamino- tathione and prevent hepatotoxicity due to overdosage glycans). with acetaminophen. When high concentrations of ace- toaminophen are present in the liver, the drug undergoes Abnormalities Involving Sulfur-Containing N-hydroxylation to form N-acetyl-benzoquinoneimine, Amino Acids which is highly reactive with sulfhydryl groups of proteins and glutathione and causes hepatic necrosis. N- Deficiencies of methionine adenosyltransferase, cys- Acetylcysteine is used as a mucolytic agent (e.g., in cystic tathionine fl-synthase, and cystathionine y-lyase have fibrosis) because it cleaves disulfide linkages of mucopro- been described. The first leads to hypermethioninemia teins. Cysteine and cystine are interconverted by NAD- but no other clinical abnormality. The second leads to hy- dependent cystine reductase and nonenzymatically by an permethioninemia, hyperhomocysteinemia, and homo- appropriate redox agent (e.g., GSH). cystinuria. The disorder is transmitted as an autosomal The major end products of cysteine catabolism in hu- recessive trait. Its clinical manifestations may include mans are inorganic sulfate, taurine, and pyruvate. Taurine skeletal abnormalities, mental retardation, ectopia lentis is a/3-amino acid that has a sulfonic acid instead of a (lens dislocation), malar flush, and susceptibility to arte- rial and venous thromboembolism. Some patients show reduction in plasma methionine and homocysteine con- L-Methionine centrations and in urinary homocysteine excretion after M~tATP hionine adenosyltransferase large doses of pyridoxine. Homocystinuria can also result PPI + P0 from a deficiency of cobalamin (vitamin BI2) or folate S-Adenosylmethionine metabolism. The third, an autosomal recessive trait, leads to cystathioninuria and no other characteristic clinical [~,~. (n CH3)in methyltransferase reaction abnormality. S-Adenosylhomocysteine Hereditary sulfite oxidase deficiency can occur alone nosylhomocysteinase or with xanthine oxidase deficiency. Both enzymes con- denosine tain molybdenum (Chapter 27). Patients with sulfite ox- HS~CH~-'CHi--~H-- COO- idase deficiency exhibit mental retardation, major motor NH~ . seizures, cerebral atrophy, and lens dislocation. Dietary L -Hornocysteine NH+ deficiency of molybdenum (Chapter 37) can cause defi- I/,-.- HOH,C----*CH--COO- L-Serine cient activity of sulfite and xanthine oxidases. ~Cystathionine [~-synthase (pyridoxal phosphate) L"--H,O Cystinuria is a disorder of renal and gastrointestinal ~H* tract amino acid transport that also affects lysine, or- * [. . OOC--CHmCl-l=w S--CHi---Cl-.l=m CH~ C OO- nithine, and arginine. The four amino acids share a com- I mon transport mechanism (discussed above). Clinically, it NH+ L -Cystathionine presents as urinary stone disease because of the insolubil- H,O ~Cystathionine ~'-Iyase (pyridoxal phosphate) ity of cystine. In cystinosis, cystine crystals are deposited ["---. NH+ (cystathionase) in tissues because of a transport defect in ATP-dependent NH+ 0 cystine efflux from lysosomes (discussed above). II OOCmCH--CH/-'-SH + CH3----CH~---~_,mCOO---.,-~--~ Succinyl-OoA L-Cysteine or Homocysteine FIGURE 17-16 Biosynthesis of cysteine. The sulfur is derived from methionine, and the Homocysteine is an amino acid not found in pro- carbon skeleton and amino group are derived from serine. teins. Its metabolism involves two pathways; one is SECTION 17.3 Metabolism of Some Individual Amino Acids 355

NH2

N ~ Sulfate O O

- - adenyl~transferase.- II II O---S--O ''"~';'~"g~---'" - O-- IIS'--O-- P--O--CH,/O oli ATP PP, O O]- I~ I I HO OH Adenosine 5"-phosphosulfate Adenylylsulfate~. ATP kinase~Mg''~ z/ "----*ADP NH=

\N~N J O O II II -O--S--O--P--O--CH2~O II I_ P~

I o OH I O--P--O- I O- PAPS FIGURE 17-17 Formation of 3'-phosphoadenosine-5'-phosphosulfate(PAPS).

the methylation of homocysteine to methionine using #-synthase have severe hyperhomocysteinemia (plasma NS-methyltetrahydrofolate (NS-methyl-FH4) catalyzed by concentrations >50 #M/L) and their clinical manifes- a vitamin B12-dependent enzyme. The second is the tations are premature atherosclerosis, thromboembolic transsulfuration pathway where homocysteine condenses complications, skeletal abnormalities, ectopia lentis and with serine to form cystathionine; this is catalyzed by mental retardation. cystathionine #-synthase (CBS) which is a pyridoxal- In plasma, homocysteine is present as both free (< 1%) 5'-phosphate enzyme. End products of the transsul- and oxidized forms (>99%). The oxidized forms include furation pathway are cysteine, taurine, and sulfate protein (primarily albumin)-bound homocysteine mixed (Figure 17-18). The methyl donor NS-methyl-FH4 is syn- disulfide (80-90%), homocysteine-cysteine mixed disul- thesized from NS,Nl~ and the reaction is fide (5-10%), and homocystine (5-10%). Several studies catalyzed by NS,Nl~ reductase have shown the relationship between homocysteine and (MTHFR). MTHFR is a FAD-dependent enzyme. Thus, altered endothelial cell function leading to thrombosis. the metabolism of homocysteine involves four water sol- Thus, hyperhomocysteinemia appears to be an indepen- uble vitamins, folate, vitamin B12, pyridoxine, and ri- dent risk factor for occlusive vascular disease. Five to ten boflavin. Any deficiencies or impairment in the conversion percent of the general population have mild hyperhomo- of the four vitamins to their active coenzyme forms will cysteinemia. affect homocysteine levels. Severe cases of hyperhomo- It has been shown that a thermolabile form of MTHFR cysteinemia occur due to deficiencies of enzymes in the is a major cause of mildly elevated plasma homocys- homocysteine remethylation or transsulfuration pathways. teine levels, which have been associated with coronary Individuals with a homozygous defect in cystathionine heart disease. The thermolabile MTHFR gene has a 3,~ CHAPTER 17 Protein and Amino Acid Metabolism

Folate (F) /i--2 NADPH+2H+ ~ ihydrofolate reductase (DR) ~ "~ 2 NADP+ Tetrahydrofolate (FH4) Methyl S-adenosylmethionine ~ (acceptors ~nfe Serine e hydroxymethyl /~ PP/+ Pi rase oxal phosphate) //( Methionine I\ / \Adenosyltransferase Methylated .i/ \ ~ Glycine ( \ATP accept~ 4~'- 1 Methylene FH4 Methionine Methylation S-adeno sylhomocy steine MTHFR (FAD) ~(~t Cycle I ethyl transferase Methyl FH4 Adenosine enosyl- ~ homocysteinase ylco~alamiHimocysteineh r gerine fysti~tOhi xO~ipnheSspy~tathea)se Cystathionine Transulfuration Pathway ~ Cystathionine 3'-lyase (pyridoxal phosphate) c~-Ketobutyrate Cysteine / \ / \ Glutathione Taurine ,v_ -~ Sulfate FIGURE 17-18 Homocysteine metabolism.

mutation of C to T at nucleotide position 677 which further studies are required to assess the utility of vitamin causes an alanine-to-valine amino acid substitution in the supplementation. protein. The mechanism by which homocysteine mediates vascular pathology remains to be understood. The Phenylalanine and Tyrosine targets for homocysteine damage are connective tissue, endothelial cells, smooth muscle cells, coagulation fac- Phenylalanine is an essential amino acid. Tyrosine is syn- tors, nitric oxide metabolism, plasma lipids and their thesized by hydroxylation of phenylalanine and there- oxidized forms (Chapter 20). Vitamin supplementation fore is not essential. However, if the hydroxylase sys- with B12, folate, and B6 has reduced total plasma homo- tem is deficient or absent, the tyrosine requirement must cysteine levels. Vitamin supplementation may decrease be met from the diet. These amino acids are involved in the morbidity and mortality from atherosclerotic vas- synthesis of a variety of important compounds, includ- cular disease due to hyperhomocysteinemia. However, ing thyroxine, melanin, norepinephrine, and epinephrine SECTION 17.3 Metabolism of Some Individual Amino Acids 357

DIET NH: J ",,,, __ ?H: < H-COO- ~ HO< >CH/---CH-- CO0-

Phenylalanine Tyrosine (essential (essential) in the event of inadequate phenylalanine supply)

protein Oxidation (liver) Thyroxine (thyroid gland) Melanin (skin pigment) Neurotransmitters and Norepinephrine adrenal medullary hormones Epinephrine+ FIGURE 17-19 Overview of the metabolism of phenylalanine and tyrosine.

(Figure 17-19). The conversion of phenylalanine to tyro- reductase is distributed widely in tissues (e.g., brain, sine and its degradation to acetoacetate and fumarate are adrenal medulla). shown in Figure 17-20. Human liver phenylalanine hydroxylase is a multimeric The phenylalanine hydroxylase reaction is complex, homopolymer whose catalytic activity is enhanced by occuring principally in liver but also in kidney. The hy- phenylalanine and has a feed-forward metabolic effect. droxylating system is present in hepatocyte cytosol and Phosphorylation of phenylalanine hydroxylase by cAMP- contains phenylalanine hydroxylase, dihydropteridine re- dependant kinase leads to increased enzyme activity and ductase, and tetrahydrobiopterin as coenzyme. The hy- dephosphorylation has an opposite effect. Thus, glucagon droxylation is physiologically irreversible and consists of and insulin have opposing effects on the catalytic activity a coupled oxidation of phenylalanine to tyrosine and of of phenylalanine hydroxylase. tetrahydrobiopterin to a quinonoid dihydroderivative with Quinonoid dihydrobiopterin is an extremely unstable molecular oxygen as the electron acceptor: compound that can rapidly rearrange (by tautomerization) phenylalanine hydroxylase to 7,8-dihydrobiopterin (Figure 17-21) and be reduced to Phenylalanine + 02 -]- tetrahydrobiopterin > the tetrahydro form by dihydrofolate reductase: tyrosine + H20 + quinonoid-dihydrobiopterin dihydrofolate reductase The tetrahydrobiopterin is regenerated by reduction of the 7,8-Dihydrobiopterin + NADPH + H + > quinonoid dihydrobiopterin in the presence of NAD(P)H NADP + + tetrahydrobiopterin by dihydropteridine reductase: This enzyme also catalyzes conversion of dihydrofolate Quinonoid dihydrobiopterin + NAD(P)H + (FH2) to tetrahydrofolate (FH4), and folic acid contains a dihydropteridine reductase pteridine ring system (see the discussion of one-carbon H + >NAD(P) + + tetrahydrobiopterin metabolism in Chapter 27). However, regeneration of NADH exhibits a lower Km and higher Vma• for tetrahydrobiopterin by the dihydrofolate reductase reac- the reductase than NADPH. Thus, the pterin coen- tion, however, is too slow to support normal rates of pheny- zyme functions stoichiometrically (in the hydroxylase lalanine hydroxylation. reaction) and catalytically (in the reductase reaction). Tetrahydrobiopterin is synthesized starting from GTP Deficiency of dihydropteridine reductase causes a and requires at least three enzymes. The first committed substantial decrease in the rate of phenylalanine hy- step is GTP-cyclohydrolase, which converts GTP to dihy- droxylation. Dihydropteridine reductase and tetrahydro- droneopterin triphosphate. 6-Pyruvoyltetrahydrobiopterin biopterin are involved in hydroxylation of tyrosine and synthase transforms dihydroneopterin triphosphate of tryptophan to yield neurotransmitters and hormones into 6-pyruvoyltetrahydrobiopterin. The latter is reduced (dopamine, norepinephrine, epinephrine, and serotonin). to tetrahydrobiopterin by NADPH-dependent sepi- Unlike phenylalanine hydroxylase, dihydropteridine apterin reductase. Deficiency of GTP-cyclohydrolase and 358 CHAPTER17 Protein and Amino Acid Metabolism

?H+ O2 Phenylalaninej20 hydroxylase CHs OH-- COO- /f ,., ~ - HO CH2-'---CH-- COO- Tetrahydro- Quinonoid- Phenylalanine biopterin . dihydrobiopterin Tyrosine NAD(P}" NAD(P)H+H+ r[ ,.> Jl [f~(z -Ketoglutarate I Dihydr0pteridine I [Tyrosine transaminase Dihydrofolate reductase pNADP* i ~"NADPH + H+ I o L_..7,8.Dihydrobiopterin ~__~ HO~ CH2--.-C--II COO - t * p-Hydroxyphenylpyruvate

Hydroxylation,shift p~p-- HyC~roxyphe nylpyruvic of the side chain I acid oxidase; ascorbic decarboxylation~.~d~CoU'+

HO~OH

COOm CH= I I CH2CO0 CH C--O II I Homogentisicacid HC CH= fO, I I Homogentisicacid CO0 CO0 oxidase; ascorbic Fumarate Acetoacetate acid; GSH H20 [ Fumarylacetoacetase m OOC H COO- \c / i C ?H2 CH.. MaleylacetoacetateHC CHz .CH, H/ \C \C / \CO0- isomerase \C / \C / \CO0- 11 II II It 0 0 0 0 Fumarylacetoacetate Maleylacetoacetate

FIGURE 17-20 Conversion of phenylalanine to tyrosine and the oxidative pathway of tyrosine.

6-pyruvoyl tetrahydrobiopterin synthase leads to hyper- (types II and III). Many mutations of the phenylalanine hy- phenylalaninemia. droxylase gene have been identified (missense, nonsense, insertions, deletions, and duplications) leading to PKU or non-PKU hyperphenylalaninemia. Phenylketonuria (PKU) The incidence of classic PKU is about 1 in 10,000- Deficiency of phenylalanine hydroxylase, tetrahy- 20,000 live births and exhibits considerable geographic drobiopterin, or dihydropteridine reductase results in variation (the incidence in Ireland is 1 in 4000, whereas phenylketonuria (PKU), an autosomal recessive trait. Be- the condition is rare among blacks and Asians). About cause phenylalanine accumulates in tissues and plasma 2% of hyperphenylalaninemic infants have a deficiency (hyperphenylalaninemia), it is metabolized by alternative of biopterin or biopterin reductase. The most important pathways and abnormal amounts of phenylpyruvate ap- clinical presentation is mental impairment. Diagnosis can pear in urine (Figure 17-22). Phenylalanine hydroxylase be made early in the neonatal period by measurement deficiency may be complete (classic PKU, type I) or partial of phenylalanine concentration in blood collected from SECTION 17.3 Metabolism of Some Individual Amino Acids 359

H tinuation throughout the first decade, or for life, may be I H~ jN~ jN~,../NH2 necessary. H/~]'~ 8IT "T Treatment of biopterin and biopterin reductase defi- H .~ II ciency consists not only of regulating the blood levels of H3C~ ?~C / "N" "hl" / phenylalanine but of supplying the missing form of coen- OH OH H zyme and the precursors of neurotransmitters, namely, ! R-group dihydroxyphenylalanine and 5-hydroxytryptophan, along 5,6,7,8-Tetrahydrobiopterin with a compound that inhibits peripheral aromatic decarboxylation. This compound is necessary because the H amine products do not cross the blood-brain barrier. I H~N~..-N~I,./NH2 H~,,- N".-,,~ N"-,~ NH Successfully treated females who have reached repro- ductive age may expose their offspring (who are obli- gate heterozygotes) to abnormal embryonic and fetal de- O O velopment. These effects include spontaneous abortion, Para-quinonoid Ortho-quinonoid microcephaly, congenital heart disease, and intrauterine dihydropteridine dihydropteridine growth retardation, and they correlate with the plasma "Quinonoid" dihydrobiopterins level of phenylalanine of the pregnant mother. Thus, H reinstitution of a low-phenylalanine diet during pre- I and postconception periods may be necessary. The diet !~i I~'~NHN~NH2 should also restrict intake of phenylalanine-containing substances, such as the synthetic sweetener aspartame (L- O aspartyl-L-phenylalanyl methyl ester). Because defective myelination occurs in the brain in PKU, there is an in- 7.8-Dihydrobiopterin creased incidence of epileptic seizures and abnormal elec- FIGURE 17-21 troencephalograms are common. The biochemical basis Structures of biopterin derivatives. for the severe mental impairment is not understood. One factor may be inhibition of glutamate decarboxylase by a heel prick onto filter paper. Treatment of phenylalanine phenylpyruvate and phenylacetate: hydroxylase deficiency consists of a diet low in phenylalanine but which maintains normal nutrition. This diet NH3 + I Glutamate decarboxylase is effective in preventing mental retardation, and its con- - OOC--(CH2)2--CH--COO- CO2 L-Glutamate ~H+ Transaminase @ 0 CHu--CH-- CO (pyridoxalphosphate) II - + O~ y,.-- ~ " CH;---C-- COO- OOC--(CH2)3--NH 3 -Ketoglutarate Glutamate Phenylalanine ~ Phenylpyruvate 7-Aminobutyrate NAD+ ~ NADH+ H+ ~ NAD+ The substrate is an excitatory neurotransmitter and the product an inhibitory one in the central nervous system." ~'~ CH2COO- C02 Abnormal indole derivatives in the urine and low OH levels of serotonin (a product of tryptophan metabolism) in Phenylacetate Phenyllactate / blood and brain point to a defect in tryptophan metabolism In the i"'-- Glutamine liver~...--H20 in PKU. 5-Hydroxytryptophan decarboxylase, which catalyzes the conversion of 5-hydroxytryptophan to 0 H COO- /! \\ II , I serotonin, is inhibited in vitro by some of the metabolites ~k__/~---OH2---C--N--(~H of phenylalanine. Phenylalanine hydroxylase is similar to the enzyme that catalyzes the hydroxylation of tryptophan to 5-hydroxytryptophan, a precursor of serotonin. In vitro, CONH2 Phenylaeelyk:jlutamine phenylalanine is also found to inhibit the hydroxylation of tryptophan. The mental defects associated with PKU may FIGURE 17-22 Formation of metabolites of phenylalanine that accumulate in abnormal be caused by decreased production of serotonin. High amounts and are excreted in phenylketonuria. phenylalanine levels may disturb the transport of amino 360 CHAPTER17 Proteinand Amino Acid Metabolism acids into cells. Variations in the clinical manifestations of PKU may reflect differences in this disturbance. Defects ~'~--C--COO- Tyrosinase --CO0- in pigmentation of skin and hair (light skin and hair) [O1 H,O H N/ may be caused by interference of melanin formation by Tyrosine 3,4-Dihydroxyphenylalanine phenylalanine and its metabolites and also by lack of (dopa) tyrosine. [O]-'~ Tyrosinase H.O~ 1 Melanin Melanin is an insoluble, high-molecular-weight poly- mer of 5,6-dihydroxyindole, which is synthesized from CO0_---.,---,,---O.,j,~~.,j+I~N/I I H--C--CO0- tyrosine (Figure 17-23). It is produced by pigment cells H (melanocytes) in cytoplasmic organelles (melanosomes). Dopachrome Dopa quinone In the epidermis, melanocytes are associated with keratinocytes, which contain melanosomes supplied by co2J zn'+ melanocytes via dendritic processes. Color variation in human skin reflects the amount of melanin synthesized in melanosomes. Melanin synthesis is apparently under hormonal and neural regulation. H H The first two steps in the synthesis of melanin are 5,6-Dihydroxy indole Indole-5,6-quinone catalyzed by tyrosinase, a copper-containing oxidase, Cysteine,,...... ,,I.- [ which converts tyrosine to dopaquinone. All subse- Trichromes 1 quent reactions presumably occur through nonenzymatic / Polymerization [ auto-oxidation, in the presence of zinc, with forma- / Pheomelanins H tion of the black to brown pigment eumelanin. The N O yellow to reddish brown, high-molecular-weight poly- mer known as pheomelanin and the low-molecular-weight trichromes result from addition of cysteine to dopaquinone and further modification of the products. Pheome- lanins and trichromes are primarily present in hair and feathers. Abnormalities of Tyrosine Metabolism N~c 0 Hepatic cytosolic tyrosine aminotransferase (tyrosine transaminase) deficiency produces tyrosinemia type II, Eumelanins an autosomal recessive trait marked by hypertyrosine- FI(;URE 17-23 mia and tyrosinuria. Clinical manifestations may include Biosynthesis of melanins. corneal erosions and plaques, inflammation (from intracellular crystallization of tyrosine), and mental retardation. Low-tyrosine and low-phenylalanine diets are beneficial. homogentisic acid. The biochemical lesion is homogen- Tyrosinosis is presumably due to fumarylacetoacetate tisic acid oxidase deficiency. Clinical features include hydrolase deficiency and has a high prevalence in the pigmentation of cartilage and other connective tissues French-Canadian population of Qu6bec. It is associ- (ochronosis) later in life from deposition of oxidized ho- ated with abnormal liver function, renal tubular dys- mogentisic acid. Patients nearly always develop arthritis function, anemia, and vitamin D-resistant rickets. Tran- in later years, but the relationship between pigment depo- sient tyrosinemia of the newborn, particularly in prema- sition and arthritis is not understood. ture infants, is the most common form of tyrosinemia in Lack of melanin production (hypomelanosis) gives rise infancy. to several hereditary disorders collectively known as al- Alcaptonuria is a rare metabolic hereditary disease in binism. Some forms result from deficiency of tyrosinase. which homogentisic acid is eliminated in urine, which The inheritance pattern of albinism varies with type. Af- darkens upon exposure to air owing to oxidation of fected individuals have increased susceptibility to various SECTION17.3 Metabolism of Some Individual Amino Acids 361

O II Tryptophanpyrrolase, E~ CH;---CHmCOO- f E~IH~I~--i7 COO NIH: 02 I H N-Formylkynurenine f-H,O Formamidase Formate~ One-carbonpool

_CmCH2--'-CH--COO- Kynurenine ,~"~C-- CHs ICHmCOO "~ I'Y ] ~. hydroxylase I- II ".§ NH.~ ~ + ....3 ' 'I , o2 NADPH + H ~_, I~ k,.~ ~NH= i; ' ~ "NH2 Z" ""~eeb~, Kynurenine 3-Hydroxykynurenine ~ o/,. ~J"Ob'o Kynureninase H20 ~e/~ (pyridoxal~ f'- ~O~,o/, ph~ ~'~Alanine -" ~ OH

CO0 ~ .... NAD+ I~/,~CO0- ~NH+ OH OH Xanthurenate 3-Hydroxyanthranilate (excretedin urine)

FIGURE 17-24 Pathway for the synthesis of NAD from tryptophan. types of carcinoma (from the effect of solar radiation on ate precursor of NAD. In deficiency of pyridoxal phos- DNA). When the eyes are involved, photophobia, sub- phate, 3-hydroxykynurenine accumulates and is converted normal visual acuity, strabismus, and nystagmus may be to xanthurenate, which is excreted in urine. Thus, vitamin present. B6 deficiency can be diagnosed by measurement of urinary xanthurenate after administration of a standard dose of tryptophan (tryptophan load test). 5-Hydroxytryptamine (serotonin) is found in ente- Tryptophan rochromaffin cells, brain, and platelets. In the former two, Tryptophan is an essential amino acid involved in it is produced from tryptophan, whereas in platelets, sero- synthesis of several important compounds. Nicotinic tonin is taken up from plasma. Synthesis involves hydrox- acid (amide), a vitamin required in the synthesis of ylation of tryptophan by tryptophan 5-hydroxylase and NAD + and NADP +, can be synthesized from tryptophan decarboxylation by aromatic L-amino acid decarboxylase (Figure 17-24). About 60 mg of tryptophan can give rise to (Figure 17-25). Hydroxylation is the rate-limiting reac- 1 mg of nicotinamide. The synthesis begins with conver- tion, is analogous to that of phenylalanine, and requires sion of tryptophan to N-formylkynurenine by tryptophan molecular oxygen and tetrahydrobiopterin. Serotonin is a pyrrolase, an inducible iron-porphyrin enzyme of liver. powerful vasoconstrictor and stimulator of smooth mus- N-Formylkynurenine is converted to kynurenine by re- cle contraction. In the brain it is a neurotransmitter, and moval of formate, which enters the one-carbon pool. in the pineal gland it serves as a precursor of melatonin. Kynurenine is hydroxylated to 3-hydroxykynurenine, Synthesis of melatonin requires N-acetylation of sero- which is converted to 3-hydroxyanthranilate, catalyzed tonin, followed by methylation (Figure 17-26). The role by kynureninase, a pyridoxal phosphate-dependent en- of melatonin in humans is not understood; in frogs, it zyme. 3-Hydroxyanthranilate is then converted by a lightens the color of skin melanocytes and blocks the series of reactions to nicotinamide ribotide, the immedi- action of melanocyte-stimulating hormone (MSH) and 362 CHAPTER17 Protein and Amino Acid Metabolism

~H;~ adrenocorticotropic hormone (ACTH). In rats, melat0nin (.CH CH--COO- regulates the breeding cycle, and its secretion is increased by exposure to light via adrenergic stimulation of the pineal gland. I N-Acetyltransferase is activated by increased concen- H Tryptophan trations of cytosolic cyclic AMP and Ca 2+ that is medi- 02, tetrahydrobiopterin~1 ated by the activation of adrenergic receptors of the pineal H20, dihydrobiopterin..~Tryptophan-5-hydroxylase gland. In humans, melatonin synthesis and its release follows a circadian rhythm which is stimulated by dark- HO~,~CHr--CH--COO - ness and inhibited by light. The blood levels of melatonin increase by passive diffusion from the central nervous system after the onset of darkness, reaching a peak value dur- I ing the middle of the night and declining during the second N 5-Hydrox~ryptophan half of the night. Melatonin secretion is also regulated en- Aromatic L-aminoacid dogenously by signals from the suprachiasmatic nucleus. r,~ .,.,~decarboxylase Since melatonin's peak concentration in plasma coincides "'=t(pydd~ phosphate) with sleep, exogenous administration of the hormone can HO. I affect the circadian rhythm. Melatonin supplementation has been used to ameliorate subjective and objective symptoms of jet lag caused by travel across time zones. At high levels, melatonin promotes sleep. Short-term and long- I N term biological effects of melatonin supplementation have 5-Hydroxytryptamine yet to be determined. (serotonin) Serotonin is degraded to 5-hydroxyindoleacetic acid FIGURE 17-25 (5-HIAA) by monoamine oxidase and aldehyde dehy- Biosynthesis of serotonin from tryptophan. drogenase acting in sequence. 5-HIAA is excreted in the urine. Its excretion is markedly increased in subjects with carcinoid tumor (found most frequently in the gastrointestinal tract and lungs). Carcinoid tumors are fre- HO.~ II--CHE--CHs NH+ quently indolent and asymptomatic; however, a signifi- jlJ Serotonin cant number of these tumors can manifest as metabolic ? problems. Although increased production of serotonin H is a characteristic feature of the carcinoid tumor, cells AcelyI'C~ 4 5-Hydrox~ryptamine- also synthesize other substances. These include kinins, CoASH~'~ N-acetyltransferase O prostaglandins, substance P, gastrin, somatostatin, corti- HO~ II cotropin, and neuron-specific enolase. Carcinoid tumor CHT'-CHu--NH~C~CH 3 cells are known as enterochromaffin cells because they N-Acetyl-5- hydroxyt ry pta m in e stain with potassium dichromate and also are known as argentaffin cells because they take up and reduce silver H salts. The symptoms of the carcinoid tumor are due to syn- ergistic biochemical interactions between serotonin and S-Adenosylmethionine-~ S.Adenosylhomocysteine.~,.[Hydroxyindole methyltransferase the above-mentioned active metabolites. Characteristics O of the carcinoid syndrome are flushing, diarrhea, wheez- ing, heart valve dysfunction, and pellagra. Lifestyle con- CHE---CHs j " ditions that precipitate symptoms include intake of alcohol or spicy foods, and strenuous exercise. The treatment N I options for the carcinoid syndrome are multidisciplinary, H including lifestyle changes, inhibitors of serotonin re- Melatonin (N-acetyl-5-methoxytryptamine) lease and serotonin receptor antagonists, somatostatin ana- FIGURE 17-26 logues, hepatic artery embolization, chemotherapy, and Biosynthesis of melatonin from serotonin. surgery. Supplemental Readings and References 363

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