Amino acid metabolism
Amino acids, in addition to their role as protein monomeric units, are energy metabolites and precursors of many biologically important nitrogen-containing compounds, notably heme, physiologically active amines, glutathione, nucleotides, and nucleotide coenzymes. Amino acids are classified into two groups: Essential and nonessential. Mammals synthesize the nonessential amino acids from metabolic precursors but must obtain the essential amino acids from their diet. Excess dietary amino acids are neither stored for future use nor excreted. Rather, they are converted to common metabolic intermediates such as pyruvate, oxaloacetate, and a-ketoglutarate. Consequently, amino acids are also precursors of glucose, fatty acids, and ketone bodies and are therefore metabolic fuels.
Amino Acid Deamination
The first reaction in the breakdown of an amino acid is almost always removal of its α-amino group with the object of excreting excess nitrogen and degrading the remaining carbon skeleton.
Most amino acids are deanimated by transamination, the transfer of their amino group to an α-keto acid to yield the α-keto acid of the original amino acid and a new amino acid, in reactions catalyized by aminotransferases. The predominant amino group acceptor is α-ketoglutarate, producing glutamate as the new amino acid:
Amino acid + α-ketoglutarate produces α-keto acid + glutamate
Glutamate's amino group, in turn, is transfered to oxaloacetate in a second transamination reaction, yielding aspartate:
Glutamate + oxaloacetate produces α-ketoglutarate + aspartate
Oxidative Deamination: Glutamate Dehydrogenase
Transamination does not result in any net deamination. Desmination occurs largely through the oxidative deamination of glutamate by glutamate dehydrogenase, yielding ammonia. The reaction requires NAD+ or NADP+ as an oxidizing agent and regenerates α-ketoglutarate for use in additional transamination reactions:
Glutamate + NAD(P)+ + H2O produces α-ketoglutarate + NH4+ + NAD(P)H
Glutamate is oxidatively deaminated in the mitochondrion by glutamate dehydrogenase, the only known enzyme that, at least in some organisms, can accept either NAD+ or NADP+ as its redox coenzyme. Oxidation is thought to occur with transfer of hydride ion from glutamate's Cα to NAD(P)+, thereby forming α-iminoglutarate, which is hydrolyzed to α-ketoglutarate and ammonia.
Other Deamination Mechanisms
Two nonspecific amino acid oxidases, L-amino acid oxidase and D-amino acid oxidase, catalyze the oxidation of the L- and D-amino acids, utilizing FAD as their redox coenzyme. The resulting FADH2 is reoxidized by O2.
Amino acid + FAD + H2O → α-keto acid + NH3 + FADH2
FADH2 + O2 → FAD + H2O2
D-amino acid oxidase occurs mainly in the kidney. Its function is an enigma since D-amino acids are associated mostly with bacterial cell walls. A few amino acids, such as serine and histidine, are deaminated nonoxidatively.
The Urea Cycle
Living organisms excrete the excess nitrogen resulting from the metabolic breakdown of amino acids in one of three ways. Many aquatic animals simply excrete ammonia. Where water is less plentiful, however, processes have evolved that convert ammonia to less toxic waste products that therefore require less water for excretion. One such product is urea, which is excreted by most terrestrial vertebrates; another is uric acid, which is excreted by birds and terrestrial reptiles.
Accordingly, living organisms are classified as being either ammonotelic (ammonia excreting), ureotelic (urea excreting), or uricotelic (uric acid excreting). Some animals can shift from ammonotelism to ureotelism or uricotelism if their water supply becomes restricted.
Urea is synthesized in the liver by the enzymes of the urea cycle. It is then secreted into the bloodstream and sequestered by the kidneys for excretion in the urine. The urea cycle was elucidated in outline in 1932 by Hans Krebs and Kurt Henseleit. Its individual reactions were later described in detail by Sarah Ratner and Philip Cohen. The overall urea cycle reaction is:
- NH3 + HCO3- + Aspartate + 3ATPs → Urea + Fumarate + 2ADPs + 2P + AMP + PP
Thus, the two urea nitrogen atoms are contributed by ammonia and aspartate, whereas the carbon atom comes from HCO3-. Five enzymatic reactions are involved in the urea cycle, two of which are mitochondrial and three cytosolic.
Carbamoyl Phosphate Synthetase: Acquisition of the First Urea Nitrogen Atom
Carbamoyl phosphate synthetase (CPS) is technically not a member of the urea cycle. It catalyzes the condensation and activation of NH4+ and HCO3- to form carbamoyl phosphate, the first of the cycle's two nitrogen-containing substrates, with the concomitant hydrolysis of two ATPs. Eukaryotes have two forms of CPS:
- Mitochondrial CPS I uses ammonia as its nitrogen donor and participates in urea biosynthesis.
- Cytosolic CPS II uses glutamine as its nitrogen donor and is involved in pyrimidine biosynthesis.
The reaction catalyzed by CPS I is thought to involve three steps:
- Activation of HCO3- by ATP to form carbonyl phosphate and ADP.
- Attack of ammonia on carbonyl phosphate, displacing the phosphate to form carbamate and P.
- Phosphorylation of carbamate by the second ATP to form carbamoyl phosphate and ADP.
The reaction is essentially irreversible and is the rate limiting step of the urea cycle. CPS I is also subject to allosteric activation by N-acetylglutamate.
Ornithine transcarbamoylase transfers the carbamoyl group of carbamoyl phosphate to ornithine, yielding citrulline. The reaction occurs in the mitochondrion so that ornithine, which is produced in the cytosol, must enter the mitochondrion via a specific transport system. Likewise, since the remaining urea cycle reactions occur in the cytosol, citrulline must be exported from the mitochondrion.
Argininosuccinare Synthetase: Acquisition of the Second Urea Nitrogen Atom
Urea's second nitrogen atom is introduced in the urea cycle's third reaction by condensation of citrulline's ureido group with an aspartate amino group by argininosuccinate synthetase. The ureido oxygen atom is activated as a leaving group through formation of a citrullyl-AMP intermediate, which is subsequently displaced by the aspartate amino group. Support for the existence of the citrullyl-AMP intermediate comes from experiments using 18O-labeled citrulline. The label was isolated in the AMP produced by the reaction, demonstrating that at some stage of the reaction, AMP and citrulline are linked covalently through the ureido oxygen atom.
With formation of argininosuccinate, all of the urea molecule components have been assembled. However, the amino group donated by aspartate is still attached to the aspartate carbon skeleton. This situation is remedied by argininosuccinase-catalyzed elimination of arginine from the aspartate carbon skeleton forming fumarate. Arginine is urea's immediate precursor. Note that the fumarate produced in the argininosuccinase reaction can be reconverted to aspartate for use in the argininosuccinate synthetase reaction. This occurs via fumarase and malate dehydrogenase reactions to form oxaloacetate followed by transamination. These are the same reactions that occur in the citric acid cycle, although they take place in the cytosol rather than in the mitochondrion.
The urea cycle's fifth and final reaction is the arginase-catalyzed hydrolysis of arginine to yield urea and regenerate ornithine. Ornithine is then returned to the mitochondrion for another round of the cycle. The urea cycle thereby converts two amino groups, one from ammonia and one from aspartate, and a carbon atom from HCO3- to the realtively nontoxic excretion product, urea, at the cost of four "high-energy" phosphate bonds (three ATP hydrolyzed to two ADP, two Pi, AMP, and PPi, followed by rapid PPi hydrolysis). This energetic cost is more than recovered however, by the energy released upon the formation of urea cycle substrates. The ammonia released by the glutamate dehydrogenase reaction is accompanied by NADH formation, as is the reconversion of fumarate through oxaloacetate to aspartate. Mitochondrial reoxidation of this NADH yields six ATPs.
Regulation of the Urea Cycle
Carbamoyl phosphate synthetase I, the mitochondrial enzyme that catalyzes the first committed step of the urea cycle, is allosterically activated by N-acetylgluamate. This metabolite is synthesized from glutamate and acetylCoA by N-acetylglutamate synthase and hydroliyzed by a specific hydrolase. The rate of urea production by the liver is, in fact, correlated with the N-acetylglutamate concentration. Increased urea synthesis is required when amino acid breakdown rates increase, generating excess nitrogen that must be excreted. Increases in these breakdown rates are signaled by an increase in glutamate concentration through transamination reactions. This situation, in turn, causes an increase in N-acetylglutamate synthesis stimulating carbamoyl phosphate synthetase and thus the entire urea cycle.
The remaining enzymes of the urea cycle are controlled by the concentrations of their substrates. Thus, inherited deficiencies in urea cycle enzymes other than arginase do not result in significant decreases in urea production (the total lack of any urea cycle enzyme results in death shortly after birth). Rather, the deficient enzyme’s substrate builds up, increasing the rate of the deficient reaction to normal. The anomalous substrate buildup is not without cost, however. The substrate concentrations become elevated all the way back up the cycle to ammonia, resulting in hyperammonemia (elevated levels of ammonia in the blood). Although the root cause of ammonia toxicity is not completely understood, a high ammonia concentration puts an enormous strain on the ammonia-clearing system, especially in the brain (symptoms of urea cycle enzyme deficiencies include mental retardation and lethargy). This clearing system involves glutamate dehydrogenase (working in reverse) and glutamine sysnthetase, which decrease the α-ketoglutarate and glutamate pools. The brain is most sensitive to the depletion of these pools. Depletion of α-ketoglutarate decreases the rate of the energy-generating citric acid cycle, whereas glutamate is both a neurotransmitter and a precursor to γ-aminobutyrate (GABA), another neurotransmitter.
Metabolic Breakdown of Individual Amino Acids
The degradation of amino acids converts them to citric acid cycle intermediates or their precursors so that they can be metabolized to CO2 and H2O or used in gluconeogenesis. Indeed, oxidative breakdown of amino acids typically accounts for 10 to 15% of the metabolic energy generated by animals. The 20 “standard” amino acids (the amino acids of proteins) have widely differing carbon skeletons, so their conversions to citric acid cycle intermediates follow correspondingly diverse pathways
Amino Acids Can Be Glucogenic, Ketogenic, or Both
"Standard" amino acids are degraded to one of seven metabolic intermediates: pyruvate, α-ketoglutarate, succinyl-CoA, fumarate, oxaloacetate, acetyl-CoA, or acetoacetate. The amino acids may therefore be divided into two groups based on their catabolic pathways:
- Glucogenic amino acids, whose carbon skeletons are degraded to pyruvate, α-ketoglutarate, succinyl-CoA, fumarate, or oxaloacetate and are therefore glucose precursors.
- Ketogenic amino acids, whose carbon skeletons are broken down to acetyl-CoA or acetoacetate and can thus be converted to fatty acids or ketone bodies.
For example, alanine is glucogenic because its transamination product, pyruvate, can be converted to glucose via gluconeogenesis. Leucine, on the other hand, is ketogenic; its carbon skeleton is converted to acetyl-CoA and acetoacetate. Since animals lack any metabolic pathway for the net conversion of acetyl-CoA or acetoacetate to gluconeogenic precursors, no net synthesis of carbohydrates is possible from leucine, or from lysine, the only other purely ketogenic amino acid. Isoleucine, phenylalanine, threonine, tryptophan, and tyrosine, however, are both glucogenic and ketogenic; isoleucine, for example, is broken down to succinyl-CoA and acetyl-CoA and hence is a precursor of both carbohydrates and ketone bodies. The remaining 13 amino acids are purly glucogenic.
Alanine, Cysteine,Glycine,Serine, and Threonine Are Degraded to Pyruvate
Five amino acids, alanine, cysteine, glycine, serine, and threonine, are broken down to yield pyruvate. Tryptophan should be included in this group since one of its breakdown products is alanine, which is transaminated to pyruvate.
Serine is converted to pyruvate through dehydration by serine dehydratase. This PLP-enzyme, like the aminotransferases, functions by forming a PLP-amino acid Schiff base so as to facilitate the removal of the amino acid's α-hydrogen atom. In serine dehydratase reaction, however, the Cα carbanion breaks down with the elimination of the amino acid's Cβ-OH, rather than with tautomerization, so that the substrate undergoes α, β elimination of H2O rather than deamination. The product of the dehydration, the enamine aminoacrylate, tautomerizes nonenzymatically to the corresponding imine, which spontaneously hydrolyzes to pyruvate and ammonia.
Cysteine may be converted to pyruvate via several routes in which the sulfhydryl group is released as H2S, SO32-, or SCN-.
Glycine is converted to serine by the enzyme serine hydroxymethyl transferase, another PLP-containing enzyme. This enzyme utilizes N5, N10-methylene-tetrahydrofolate (N5, N10-methylene-THF) as a cofactor to provide C1 unit necessary for this conversion. The methylene group of the N5, N10-methylene-THF utilized in conversion of glycine to serine is obtained through a second glycine degradation catalyzed by the glycine cleavage system. The glycine cleavage system, a multienzyme complex that resembles pyruvate dehydrogenase, contains four protein components:
- A PLP-dependent glycine decarboxylase (P protein).
- A lipoamide-containing aminomethyltransferase (H protein), which carries the aminomethyl group remaining after glycine decarboxylation.
- An N5, N10-methylene-THF synthesizing enzyme (T protein), which accepts a mthylene group from the aminomethyltransferase.
- An NAD+-dependent, FAD-requiring lipoamide dehydrogenase (L protein).
Two observations indicate that this pathway is the major route of glycine degradation in mammalian tissues:
- The serine isolated from an animal that has been fed [2-14C]glycine is 14C labeled at both C2 and C3. This observation indicates that the methylene group of the N5, N10-methylene-THF utilized by serine hydroxymethyl transferase is derived from glycine C2.
- The inherited human disease nonketotic hyperglycinemia, which is characterized by mental retardation and accumulation of large amounts of glycine in body fluids, results from the absence of the glycine cleavage system.
Threonine is both glucogenic and ketogenic, since one of its degradation routes produces both pyruvate and acetyl-CoA. Its major route of breakdown is through threonine dehydrogenase, producing α-amino-β-ketobutyrate, which is converted to acetyl-CoA and glycine by α-amino-β-ketobutyrate lyase. The glycine may be converted, through serine, to pyruvate.
Serine Hydroxymethyl Transferase Catalyzes PLP-Dependent Cα-Cβ bond cleavage
Threonine may also be converted directly to glycine and acetaldehyde. Surprisingly, this reaction is catalyzed by serine hydroxymethyl transferase. Biochemists have heretofore considered PLP-catalyzed reactions that begin with the cleavage of an amino acid’s Cα-Cβ bond by delocalizing the electrons of the resulting carbanion into the conjugated PLP ring.
How can the same amino acid-PLP Schiff base be involved in the cleavage of the directional bonds to an amino acid Cα in different enzymes? The answer to this conundrum was suggested by Harmon Dunathan. For electrons to be withdrawn into the conjugated ring system of PLP, the π-orbital system of the PLP must overlap with the bonding orbital containing the electron pair being delocalized. This is possible only if the bond being broken lies in the plane perpendicular to the plane of the PLP π-orbital system. Different bonds to Cα can be placed in this plane by rotation about the Cα-N bond. Indeed, the X-ray structure of aspartate aminotransferase reveals that the Cα-H of its aspartate substrate assumes just this conformation. Evidently, each enzyme specifically cleaves its corresponding bond because the enzyme binds the amino acid PLP Schiff base adduct with this bond in the plane perpendicular to that of the PLP ring. This is an example of stereoelectronic assistance: The enzyme binds substrate in a conformation that minimizes the electronic energy of the transition state.
Asparagine and Aspartate Are Degraded to Oxaloacetate
Transamination of aspartate leads directly to oxaloacetate. Asparagine is also converted to oxaloacetate in this manner after its hydrolysis to aspartate by L-asparaginase. Interestingly, L-asparaginase is an effective chemotherapeutic agent in the treatment of cancers that must obtain asparagine from the blood, particularly acute lymphoblastic leukemia. It is uncertain, however, whether cell death results from the depletion of asparagine levels in the blood or from some other metabolite of the L-asparaginase reaction.
Arginine, Glutamate, Glutamine, Histidine, and Proline Are Degraded to α-Ketoglutarate
Arginine, glutamine, histidine, and proline are all degraded by conversion to glutamate, which in turn is oxidized to α-ketoglutarate by glutamate dehydrogenase. Conversion of glutamine to glutamate involves only one reaction: hydrolysis by glutaminase. Histidine's conversion to glutamate is more complicated: It is nonoxidatively deaminated, then it is hydrated, and its imidazole ring is cleaved to form N-formiminoglutamate. The formimino group is then transferred to tetrahydrofolate forming glutamate and N5-formiminotetrahydrofolate. Both arginine and proline are converted to glutamate through the intermediate formation of glutamate-5-semialdehyde.
Isoleucine, Methionine, and Valine Are Degraded to Succinyl-CoA
Isoleucine, methionine, and valine have complex degradative pathways that all yield propionyl-CoA. Propionyl-CoA, which is also a product of odd-chain fatty acid degradation, is converted to succinyl-CoA by a series of reactions involving the participation of biotin and coenzyme B12.
Methionine Breakdown Involves Synthesis of S-adenosylmethionine and Cysteine
Methionine degradation begins with its reaction with ATP to form S-adenosylmethionine. This sulfonium ion's highly reactive methyl group makes it an important biological methylating agent. For instance, S-adenosylmethionine is the methyl donor in the synthesis of phosphatidylcholine from phosphatidylethanolamine. It is also the methyl donor in the conversion of norepinephrine to epinephrine.
Meythlation reactions involving S-adenosylmethionine yield S-adenosylhomocycteine in addition to the methylated acceptor. The former product is hydrolyzed to adenosine and homocysteine in the next reaction of the methionine degradation pathway. The homocysteine may be methylated to form methionine via a reaction in which N5-methyl-THF is the methyl donor. Alternatively, the homocysteine may combine with serine to yield cystathionine, which subsequently forms cystine and α-ketobutyrate. The α-ketobutyrate continues along the degradative pathway to propionyl-CoA and then succinyl-CoA.
Branched-Chain Amino Acid Degradation Pathways Contain Themes Common to All Acyl-CoA Oxidations
Maple Syrup Urine Disease Results from a Defect in Branched-Chain Amino Acid Degradation
Branched chain α-keto acid dehydrogenase (BCKDH; also known as α-ketoisovalerate dehydrogenase) is a multienzyme complex that closely resembles the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase multienzyme complexes. Indeed, all three of these multienzyme complexes share a common protein component, E3 (dihydrolipoamide dehydrogenase), and employ the coenzymes TPP, lipoamide, and FAD in addition to their terminal oxidizing agent, NAD+.
A genetic deficiency in BCKDH causes maple syrup urine disease, so named because the consequent buildup of branched-chain α-keto acids imparts the urine with the characteristic odor of maple syrup. Unless promptly treated by a diet low in branched-chain amino acids, maple syrup urine disease is rapidly fatal.