Metabolism of Amino Acids

Urea Cycle–Related Disease: Hyperammonemia

The urea cycle is one of the metabolic pathways that will be discussed in this chapter. The urea cycle eliminates unneeded nitrogen, derived from nitrogen-containing compounds, from the body in the form of urea. Many amino acids can be converted to glutamate that can, in turn, be converted to aspartate . Aspartate can enter the urea cycle to produce urea for excretion in the urine. If there is a defect in the urea cycle, excess ammonia (hyperammonemia) accumulates in the blood and results in devastating disease, including death. Defects in the cycle are genetic (develop in infants) and involve deficiency of carbamoylphosphate synthase and/or ornithine transcarbamylase . Other enzyme deficiencies also contribute by accumulation of the substrate for the deficient enzyme. Thus argininosuccinic acid synthase (citrulline+Asp+ATP→argininosuccinate+AMP+PP _i ) deficiency leads to citrullinuria , and argininosuccinate lyase (argininosuccinate→Arg+fumarate) deficiency causes argininosuccinic aciduria .

Normally, the conversion of amino acid–derived ammonia to urea is highly efficient and protects the central nervous system (CNS) from toxicity. Elevations in the blood level of ammonia occur in an infant when an enzyme activity is low or missing altogether from a genetic defect. In an adult, this can occur from a diseased liver, the organ site of the urea cycle but there appears to be increasing incidence of underlying genetic disease in previously normal adults. Increased blood levels of ammonia produce a variety of symptoms as shown in Fig. 13.1 .

The normal blood level of ammonia is roughly 500 nmol/L. The normal level of blood urea nitrogen is about 300 μmol/L. There is a factor of about 600-fold or more urea than ammonia. Urea cycle disorders have a frequency of about one in 30,000 in the newborn.

In the cerebellum and striatum of the brain, ammonia hyperactivates the excitatory N -methyl- d -aspartate receptor (NMDAR) at its glycine site, resulting in the accumulation of cyclic guanosine monophosphate ( cGMP ) [when Glu from a presynaptic nerve ending crosses the synapse and activates the NMDAR on the postsynaptic membrane, it leads to an influx of Ca ²⁺ which binds to calmodulin which, in turn, activates neuronal nitric oxide synthase (nNOS) to produce nitric oxide (NO). NO then crosses the synapse back to the presynaptic membrane to activate guanylate cyclase and produce cGMP from guanosine triphosphate (GTP)].

The ammonia (NH ₃ or ammonium ion, NH ₄ ⁺ ) in the brain is utilized by glutamate dehydrogenase (GDH) wherein α-ketoglutarate+ NH ₄ ⁺ is converted to glutamate. This lowering of α-ketoglutarate (and subsequently oxaloacetate) depresses the tricarboxylic acid (TCA) cycle and aerobic oxidation and leads to damage and death of the cell ( Fig. 13.2 ).

There are several urea cycle–related disorders. Most derive from genetic abnormalities involving genes for the enzymes in the urea cycle ( Table 13.1 ).

The plasma level of ammonia is determined if hyperammonemia is suspected. The condition of the liver is also tested by measuring cellular components that may leak into the bloodstream when liver cells are damaged. These component activities include serum transaminases and alkaline phosphatase . The level of serum albumin can be measured as the level can fall below 3.5 mg/dL in advanced liver disease. Prothrombin time is also measured as the proteins of the coagulation system are made in the liver. There is a good correlation between prothrombin time and liver function. Increased plasma levels of citrulline or argininosuccinic acid can be a signal for primary genetic disease. If there is a general increase in plasma amino acid levels, liver disease may be the problem. Partial inhibition of the urea cycle can be generated by dysfunctions of amino acid catabolism (to be discussed later), and this can lead to increased levels of blood ammonia. In this case, intermediates of amino acid catabolism may be increased in the blood, and these would include propionic acid , methylmalonic acid , and isovaleric acid . Amino acid profiles in the urine can characterize argininosuccinic aciduria , hyperornithinemia , hyperammonemia , homocitrullinuria , or intolerance to dibasic amino acids in dietary protein (lysinuric protein intolerance). The blood lactic acid concentration can rule out mitochondrial diseases because the failure to utilize pyruvate efficiently by the mitochondrial TCA cycle will cause an increase of lactate in the blood. The pH of blood may be elevated when blood ammonia level is increased because it stimulates the respiratory system. The normal range of blood urea nitrogen is 8–20 mg/dL, but in disorders of the urea cycle , it can be less than 3 mg/dL. The treatment of hyperammonemia requires reduced protein intake and replacing the protein with nonprotein sources. Sodium phenylacetate or Ucephan (sodium benzoate) is administered intravenously as it stimulates the excretion by the kidneys of nitrogen as phenylacetylglutamine and hippuric acid . Sodium benzoate conjugates with glycine to form hippuric acid that is also excreted by the kidneys. The combination of the two reagents ( Ammonul ) is also used. Molecular genetics (gene sequencing) confirms the diagnosis of a genetic disease of the urea cycle. If elevated levels of ammonia are not treated, there is damage to the CNS in terms of cell death; cerebral edema will follow with increased intracranial pressure, and death will result.

The Urea Cycle

Total 80% of urea is synthesized from ammonia in the liver in the urea cycle. When glutamine is produced in excess in the liver, it is converted to ammonia by a glutaminase enzyme found in the periportal hepatocytes and renal epithelial cells (the enzyme also is located in the intestine). Ammonium ion is important in acid–base regulation in the kidney, and during acidosis , this enzyme is induced in the kidney to increase the excretion of ammonium (NH ₄ ⁺ ). The glutaminase reaction is

The liver urea cycle functions by converting ammonia (as ammonium ion) to urea that is excreted in the urine. The cycle consists of five enzymes of which the initial two enzymes are located in the mitochondrial matrix, and the other three are located in the liver soluble cytoplasm. The urea cycle is also referred to as the Krebs–Henseleit cycle named after the discoverers.

Ammonia is derived from dietary amino acids and proteins (plants fix nitrogen from the atmosphere to form ammonia). Proteins are broken down in the intestinal tract and absorbed as peptides and free amino acids. These become precursors of human proteins and the excess amino acids, not needed for protein synthesis, are deaminated to produce ammonia. The deaminated products enter the TCA cycle. Ammonia (ammonium ion) from amino acid metabolism enters the mitochondrial matrix and is combined with bicarbonate and adenosine triphosphate (ATP) to form carbamoylphosphate , the first step in the urea cycle ( Fig. 13.3 ).

The first step is catalyzed by carbamoylphosphate synthase I . Ornithine transcarbamylase converts carbamoylphosphate to citrulline . These two steps take place in the mitochondrial matrix . Citrulline is transported out of the mitochondrial matrix, by a transporter, to the soluble cytoplasm. In the cytosol, citrulline is combined with aspartate and is converted to argininosuccinate by argininosuccinate synthase . Argininosuccinate is converted to fumarate and arginine by argininosuccinate lyase . Fumarate can enter the mitochondrial TCA cycle and arginine is converted by arginase , the terminal enzyme of the urea cycle, to one molecule of urea and one molecule of ornithine. Ornithine can be transported to the mitochondrial matrix by an ornithine transporter and take part in the urea cycle . The interactions between the urea cycle and the mitochondrial matrix are summarized in Fig. 13.4 .

The overall reactions of the TCA cycle and the urea cycle can be summarized as

Amino Acid Metabolism: Amino and Amide Group Transfers

As mentioned, GDH forms glutamate from α-ketoglutarate and ammonia (NH ₄ ⁺ , ammonium). Glutamate is converted to glutamine by glutamine synthase . The glutamine synthase catalyzed reaction is

Glutamine can be broken down to glutamate and ammonium ion by glutaminase :

Transamination accounts for the addition of amino groups from these amino acids to carbon skeletons to form other amino acids. The amide group from glutamine can be transferred to carbon skeletons by transamidation . GDH, in its reverse reaction, can convert glutamate into ammonium and α-ketoglutarate that can enter the TCA cycle for the production of energy and the reduced coenzymes NADH+H ⁺ or NADPH + H ⁺ (GDH can use either coenzyme). GDH is localized to the mitochondrial matrix (although some evidence suggests that the enzyme may not be entirely mitochondrial) and is a branch point that links amino acids to energy metabolism. GDH is a hexameric enzyme consisting of two stacks of trimers. It is in high concentration in liver, brain, kidney, and pancreas but is not in high concentration in muscle. It is an allosteric enzyme regulated by the positive effectors ATP and GTP directing the reaction to the formation of glutamate. The positive effectors, ADP and GDP, regulate the enzyme in the reverse direction for the formation of ammonium ion and α-ketoglutarate. The ATP concentration in the cell (the cellular level of ATP varies between 1 and 10 mM) determines the direction of the GDH reaction. Thus when [ATP] is high in the cell, the conversion of glutamate to α-ketoglutarate is depressed; the keto acid is not needed as a source of energy. However, when the concentration of ATP is low, glutamate is converted to α-ketoglutarate so that it can enter the TCA cycle for the production of ATP.

There are two forms of human GDH, GDH1 and GDH2 . The two isozymes differ in their endogenous activity, allosteric regulation, and stability to heat. The overall reaction catalyzed by GDH is

GDH1 is markedly inhibited by GTP, but GDH2 is not. GDH2 has low basal activity and can be fully activated by ADP or l -leucine. GDH2 is concentrated in testis and brain . In the testis the enzyme is localized to the Sertoli cells , while in the brain, it is localized to the astrocytes and in low concentration in neurons. Astrocytes support neurons and Sertoli cells support germ cells. Because GDH2 is not controlled by GTP, the selective expression of this isoform allows GDH2 to metabolize glutamate even when the TCA cycle is generating GTP in amounts sufficient to inactivate the GDH1 isozyme, thus allowing the supporting cells, astrocytes, and Sertoli cells, to continue functioning even when oxidative metabolism is high.

Glutamine is a major amino acid in blood that carries ammonia from various tissues; it is important in the transport of ammonia from peripheral tissues to the kidney. In the kidney, glutamine amide nitrogen is cleaved by glutaminase to produce glutamate plus ammonium ion that is excreted in the urine. In tissues that contain the urea cycle , glutaminase is also present allowing for ammonia to be incorporated either into urea or glutamine. Both urea and NH ₄ ⁺ are excreted into the urine by the kidney .

In the case of acidosis (when the blood pH becomes acidic), more glutamine is transferred from the liver to the kidney so that HCO ₃ ⁻ is conserved (in the urea cycle, two bicarbonate ions are used per ammonium ion to form urea). Kidney glutaminase releases glutamate and ammonium ion from glutamine, and GDH can release another mole of NH ₄ ⁺ (plus α-ketoglutarate) so that ammonium ion (carrying a proton) can be excreted. The net effect is a reduction of protons (H ⁺ ) causing an increase in pH to overcome acidosis.

The generation of GDH in the developing brain is essential for the control of ammonia detoxification . When its activity is low or lacking during development, mental retardation can occur because the toxic effects of ammonia cannot be dealt with effectively.

Glutamine synthase in the liver is localized to the perivenous hepatocytes (surrounding a vein other than the portal vein), whereas glutaminase is periportal (surrounding the portal vein). While glutamine is the most important transporter of nitrogen between tissues, the conversion of glutamate to glutamine occurs intracellularly. In periportal hepatocytes, glutamine is used for the synthesis of glucose : glutaminase converts glutamine to glutamate plus ammonium ion, and glutamate is converted to α-ketoglutarate, by transamination, which enters the TCA cycle and then through gluconeogenesis to produce glucose. Ammonium ion enters the urea cycle. α-Ketoglutarate is converted to malate in the TCA cycle and, when cellular concentrations of glucose are low, malate is transported to the soluble cytoplasm via the malate/pyruvate shuttle , and malate is converted to pyruvate by cytoplasmic malic enzyme-1 (malic enzyme-2 operates within the mitochondria to convert malate to pyruvate), and two molecules of pyruvate are converted to glucose in the gluconeogenic pathway (reverse direction of glycolysis).

Glutamine is formed from lactate and arginine in perivenous hepatocytes. This occurs by the conversion of lactate to α-ketoglutarate that is converted to glutamate and then to glutamine via glutamine synthase. Arginine is converted to ornithine by arginase , and ornithine is converted to pyrroline-5-carboxylate by ornithine aminotransferase , and pyrroline-5-carboxylate is converted to glutamate by 1-pyrroline-5-carboxylate dehydrogenase and then to glutamine by glutamine synthase. A summary of the events in the periportal hepatocyte compared to the perivenous hepatocyte is shown in Fig. 13.5 .

Transamination

Transamination is the process by which amino groups are removed from amino acids and transferred to acceptor keto acids to generate the amino acid version of the keto acid and the keto acid version of the original amino acid. The reactions are highly reversible, and the forward or reverse direction depends upon the concentrations of substrates or products. This class of enzymes contains pyridoxal phosphate (PLP) as coenzyme, although certain transaminases (also termed aminotransferases) can use pyruvate as in glutamate–pyruvate transaminase:

α-Ketoglutarate is sometimes written as 2-oxoglutarate .

Aspartate aminotransferase is an important enzyme across many species and catalyzes the reaction:

There are two different forms of this enzyme (different primary amino acid sequence), one residing in the mitochondrion and one in the cytosol (soluble cytoplasm). The enzyme also has been named glutamate–oxaloacetate transaminase , and the two forms have been referred to as s-GOT and m-GOT (see Chapter 3 : Introductory Discussion on Water, pH, Buffers, and General Features of Receptors, Channels, and Pumps). In Fig. 13.6 is shown the general reaction mechanism for a transaminase.

Muscle cells rely on glutamate–pyruvate transaminase to produce alanine from pyruvate and an amino acid so that the keto acid produced (like α-ketoglutarate) can be used as fuel for the TCA cycle for the production of energy as ATP. The alanine is carried to the liver in the bloodstream so that the amino groups from amino acids can be converted to urea in the urea cycle . In this way, muscle cells can use amino acids as energy sources while relying on the liver to deal with the amino groups (as ammonium ions). Alanine, a predominant amino acid in proteins, is also transported in the bloodstream to the liver where it can be converted to glucose. Transamination of alanine to pyruvate allows pyruvate to form glucose through the gluconeogenic pathway . The amino group of alanine is attached to α-ketoglutarate through transamination into glutamate. The amino group of glutamate is removed as NH ₄ ⁺ by GDH for incorporation into urea that is cleared through the kidney. These reactions are known as the alanine cycle , summarized in Fig. 13.7 .

During stress , especially prolonged stress, the adrenal cortex secretes cortisol. Even in short events of stress, there is enough cortisol secreted to increase the level of circulating glucose (~10% increase) that evokes a release of insulin. Prolonged stress results in the breakdown of the musculature releasing amino acids into the bloodstream, of which alanine is predominant because of its plentiful occurrence in many proteins. Increased [alanine] is taken up by the liver where it can be converted to glucose (alanine cycle) and, under the influence of insulin , can be converted to glycogen .

Another important enzyme is γ-aminobutyric acid ( GABA , also 4-aminobutanoic acid) transaminase. GABA is a key amino acid in the CNS, being the main inhibitory neurotransmitter. Although it is technically an amino acid, it is not incorporated into protein . It is an excitability factor, operating through GABA receptors, in the nervous system, and GABA can be acted upon by GABA transaminase :

This enzyme is one control on the GABA concentration, and GABA can be converted to a form that serves as an energy source. Moreover, the glutamate produced in the reaction also can be converted back to α-ketoglutarate (e.g., glutamate–oxaloacetate transaminase ) that can enter another round of GABA transaminase activity, or it can enter the TCA cycle to serve as an energy source.

Transamidation

Transamidation is an enzyme-catalyzed reaction. Transamidases catalyze the formation of a covalent bond between a free amine group (e.g., lysine bound to a protein or peptide) and the gamma-carboxamide group (e.g., glutamine bound to a protein or peptide). In this case the transamidase is a transglutaminase . A simple transamidase reaction is

In this reaction the substrate (1) is glutamine and the product (2) is the glutamine cross-reacted with the initial amine containing an R group. The same reaction could apply to lysine as the substrate (1). These enzymes require Ca ²⁺ that complexes substrate (1) to the enzyme. Transamidinases catalyze the formation of γ-glutamyl-ε-lysine bonds that may be involved in tissue healing ( Fig. 13.8 ).

There are eight transglutaminases (they are calcium-dependent mammalian enzymes): Factor VIII that is the fibrin-stabilizing factor in blood coagulation; three enzymes in skin: keratinocyte transglutaminase , epidermal transglutaminase , and transglutaminase MX ; a widely distributed transglutaminase, tissue transglutaminase ; transglutaminase MZ in testis and lung; prostate transglutaminase and transglutaminase MY .

In pulmonary (arterial) smooth muscle cells, serotonin enters the cell through the serotonin transporter and becomes transamidated to small GTPases, such as RhoA that activates ROCK (rho-associated kinase) .

Transamidation is important in the synthesis of aminosugars . In particular, the C5 (ε) amide of glutamine is contributed to fructose-6-phosphate to form glutamate and glucosamine-6-phosphate . Aminosugars are found in certain proteoglycans , probably on human chondroitin, but most aminosugars are found in plants and bacteria. Interestingly, there is a human testis glucosamine-6-phosphate deaminase (~33 kDa) found in human sperm that seems to show an oscillation-inducing (Ca ²⁺ oscillations) activity in human eggs.

Most important is the transamidase reaction involved in the glycosylphosphatidylinositol ( GPI ) anchoring of proteins to a cellular membrane. The C-terminal amino acid of a protein is attached to the GPI, and the protein is stably anchored to the outer leaflet of the lipid bilayer membrane. Although many proteins are anchored to a membrane by transmembrane hydrophobic polypeptides, the GPI system is the mechanism for many attachments of proteins (including the prion protein ) to a membrane. A schematic of the structure of a GPI-anchored protein and the transamidase reaction involved is shown in Fig. 13.9 .

Human GPI transamidase is a complex enzyme consisting of five subunits with portions of each transiting the endoplasmic reticulum (ER) membrane and portions extending into the lumen of the ER and other portions extending into the cellular cytosol ( Fig. 13.10 ).

It has been suggested that there is a conserved proline residue in the last transmembrane segment of GAA1 creating a hinge region that is the structural basis for the interaction of GPI precursor and protein precursor ( Fig. 13.9B ).

Deamination

Deamination of amino acids, mainly serine and threonine, is catalyzed by either serine dehydratase or threonine dehydratase (these enzymes may also be referred to as Ser or Thr deaminase, Ser or Thr dehydratase, or Ser or Thr ammonia lyase). These reactions are nonoxidative. The coenzyme for these enzymes is PLP and the reactions catalyzed are

and

Both enzymes are classified as ammonia lyases .

A more detailed reaction is shown in Fig. 13.11 .

The enzyme, histidase (histidine ammonia lyase), removes the amino group from histidine to form transurocanic acid . The enzyme is located in the liver and skin. In skin, ultraviolet (UV) light causes the isomerization of transurocanic acid into cis -urocanic acid . The reactions are shown in Fig. 13.12 .

Although the previous reactions are significant, glutamic acid is the major amino acid for deamination reactions. Other amino acids can be deaminated but these reactions occur mainly by transamination and deamination of glutamate where glutamate recycles. Thus any amino acid can react with α-ketoglutarate to form glutamate plus the keto acid analog of the original amino acid. Then, glutamate can be deaminated to produce ammonia (from the original amino acid) that is cleared through the urea cycle and excreted. Since glutamate is the product of many transamination reactions, it is the main substrate for oxidative deamination (by GDH in liver and other tissues). The GDH reaction is

NH ₃ is converted to urea through the urea cycle and excreted by the kidney. As mentioned previously, GDH is an allosteric enzyme where ADP is an activator and GTP, an inhibitor ( Fig. 13.13 ).

In the chapter on nucleic acids, deamination reactions for cytosine (to uracil), 5-methylcytosine (to thymine), guanine (to xanthine), and adenine (to hypoxanthine) have been discussed.

Oxidation of Amino Acids

Liver and kidney peroxisomes contain d -amino acid oxidase (as well as l -amino acid oxidase) that directly oxidizes d -amino acids ( d -amino acids are not incorporated into proteins). The coenzyme is flavin-adenine dinucleotide ( FAD ), and the enzyme exhibits wide specificities. An imino acid is produced with H ₂ O ₂ , and ammonia is removed from the enzyme intermediate as shown in Fig. 13.14 . Plants and bacteria do not have this enzyme, so many d -amino acids may occur in the diet. The human also has amino acid racemase that converts l -amino acids to d -amino acids.

There are small amounts of l -amino acid oxidase in the liver and kidney peroxisomes. This would come into play when there is an excess of amino acids for all other pathways.

An extensive loss of motor neurons occurs in amyotrophic lateral sclerosis ( ALS ). This may be due to aberrant excitability of motor neurons that contributes to their cell death. There is a mutation in the d -amino acid oxidase gene that causes the downregulation of serine degradation resulting in the accumulation of d -serine, and these effects are associated with familial ALS. d -Serine is a coagonist (besides glycine) of the NMDAR that causes excitability in the brain. Serine racemase is also present in the human brain, and it generates d -serine from l -serine. The expression of serine racemase is altered in some mental diseases, such as schizophrenia .

Amino Acid Racemization

High levels of d -serine in the human brain need to be regulated due to its coagonist action on NMDAR resulting in damaging excitability that can lead to cell death. Although l -amino acid racemase , the enzyme converting an l -amino acid to a d -amino acid, contributes to the metabolism of glycine, serine, threonine, and cysteine, it is important in the brain for its contribution to the level of d -serine (large amounts of d -serine occur in the hippocampus and corpus callosum ). Inside the cell, l -serine can be converted to d -serine by the racemase and d -serine can either be transported out of the cell, or it can be converted to pyruvate and ammonia by an α,β-elimination. The extracellular d -serine can form a complex with the membrane NMDAR or can be taken up again by the original cell (or transported out of the brain). This metabolism is shown in Fig. 13.15 and includes the l -serine racemase reaction mechanism as well as the mechanism of α,β-elimination to form pyruvate and ammonia (NH ₃ or NH ₄ ⁺ ).

l -Serine racemase would seem to be a good therapeutic target for some of these diseases based on high brain d -serine levels. There would have to be a site different from the coenzyme-binding site (because there are many PLP-requiring enzymes in the body, and there would be the added problem of crossing the blood–brain barrier).