Biochemistry | © Simon Sindayigaya; contributors: Franco Longhini |
We now turn our attention to the amino acids, the final class of
biomolecules that, through their oxidative degradation, make a significant
contribution to the generation of metabolic energy. The fraction of metabolic
energy obtained from amino acids, whether they are derived from dietary protein
or from tissue protein, varies greatly with the type of organism and with
metabolic conditions. Carnivores can obtain (immediately following a meal) up to
90% of their energy requirements from amino acid oxidation, whereas herbivores
may fill only a small fraction of their energy needs by this route. Most
microorganisms can scavenge amino acids from their environment and use them as
fuel when required by metabolic conditions. Plants, however, rarely if ever
oxidize amino acids to provide energy; the carbohydrate produced from CO2 and
H2O in photosynthesis is generally their sole energy source. Amino acid
concentrations in plant tissues are carefully regulated to just meet the
requirements for biosynthesis of proteins, nucleic acids, and other molecules
needed to support growth. Amino acid catabolism does occur in plants, but its
purpose is to produce metabolites for other biosynthetic pathways.
In animals, amino acids undergo oxidative degradation in three different
metabolic circumstances:
1. During the normal synthesis and degradation of cellular proteins (protein
turnover), some amino acids that are released from protein breakdown and are not
needed for new protein synthesis undergo oxidative degradation.
2. When a diet is rich in protein and the ingested amino acids exceed the body’s
needs for protein synthesis, the surplus is catabolized; amino acids cannot be
stored.
3. During starvation or in uncontrolled diabetes mellitus, when carbohydrates
are either unavailable or not properly utilized, cellular proteins are used as
fuel.
Under all these metabolic conditions, amino acids lose their amino groups to
form α-keto acids, the “carbon skeletons” of amino acids. The α-keto acids
undergo oxidation to CO2 and H2O or, often more importantly, provide three- and
four-carbon units that can be converted by gluconeogenesis into glucose, the
fuel for brain, skeletal muscle, and other tissues. The pathways of amino acid
catabolism are quite similar in most organisms. The focus of this chapter is on
the pathways in vertebrates, because these have received the most research
attention. As in carbohydrate and fatty acid catabolism, the processes of amino
acid degradation converge on the central catabolic pathways, with the carbon
skeletons of most amino acids finding their way to the citric acid cycle.
One important feature distinguishes amino acid degradation from other catabolic
processes described to this point: every amino acid contains an amino group, and
the pathways for amino acid degradation therefore include a key step in which
the -amino group is separated from the carbon skeleton and shunted into the
pathways of amino group metabolism (Fig. 14–1).
FIGURE 14–1 Overview of amino acid catabolism in mammals. The amino groups and the carbon skeleton take separate but interconnected pathways.
Nitrogen, N2, is abundant in the atmosphere but is too inert for use in most
biochemical processes. Because only a few microorganisms can convert N2 to
biologically useful forms such as NH3, amino groups are carefully husbanded in
biological systems. Figure 14–2a provides an overview of the catabolic pathways
of ammonia and amino groups in vertebrates. Amino acids derived from dietary
protein are the source of most amino groups. Most amino acids are metabolized in
the liver. Some of the ammonia generated in this process is recycled and used in
a variety of biosynthetic pathways; the excess is either excreted directly or
converted to urea or uric acid for excretion, depending on the organism (Fig.
14–2b). Excess ammonia generated in other (extrahepatic) tissues travels to the
liver (in the form of amino groups, as described below) for conversion to the
excretory form.
Glutamate and glutamine play especially critical roles in nitrogen metabolism,
acting as a kind of general collection point for amino groups. In the cytosol of
hepatocytes, amino groups from most amino acids are transferred to
α-ketoglutarate to form glutamate, which enters mitochondria and gives up its
amino group to form NH4+. Excess ammonia generated in most other tissues is
converted to the amide nitrogen of glutamine, which passes to the liver, then
into liver mitochondria. Glutamine or glutamate or both are present in higher
concentrations than other amino acids in most tissues. In skeletal muscle,
excess amino groups are generally transferred to pyruvate to form alanine,
another important molecule in the transport of amino groups to the liver. We
begin with a discussion of the breakdown of dietary proteins, then give a
general description of the metabolic fates of amino groups.
FIGURE 14–2 Amino group catabolism. (a) Overview of catabolism of amino groups (shaded) in vertebrate liver. (b) Excretory forms of nitrogen. Excess NH4 is excreted as ammonia (microbes, bony fishes), urea (most terrestrial vertebrates), or uric acid (birds and terrestrial reptiles). Notice that the carbon atoms of urea and uric acid are highly oxidized; the organism discards carbon only after extracting most of its available energy of oxidation.
In humans, the degradation of ingested proteins to their constituent amino
acids occurs in the gastrointestinal tract. Entry of dietary protein into the
stomach stimulates the gastric mucosa to secrete the hormone gastrin,
which in turn stimulates the secretion of hydrochloric acid by the parietal
cells and pepsinogen by the chief cells of the gastric glands (Fig. 14–3a). The
acidic gastric juice (pH 1.0 to 2.5) is both an antiseptic, killing most
bacteria and other foreign cells, and a denaturing agent, unfolding globular
proteins and rendering their internal peptide bonds more accessible to enzymatic
hydrolysis. Pepsinogen (Mr 40,554), an inactive precursor is converted to
active
pepsin (Mr 34,614) by the enzymatic action of pepsin itself. In the
stomach, pepsin hydrolyzes ingested proteins at peptide bonds on the
amino-terminal side of the aromatic amino acid residues Phe, Trp, and Tyr,
cleaving long polypeptide chains into a mixture of smaller peptides. As the
acidic stomach contents pass into the small intestine, the low pH triggers
secretion of the hormone
secretin into the blood. Secretin stimulates the pancreas to secrete
bicarbonate into the small intestine to neutralize the gastric HCl, increasing
the pH to about 7. (All pancreatic secretions pass into the small intestine
through the pancreatic duct.) The digestion of proteins now continues in the
small intestine. Arrival of amino acids in the upper part of the intestine
(duodenum) causes release into the blood of the hormone cholecystokinin,
which stimulates secretion of several pancreatic enzymes with activity optima at
pH 7 to 8.
Trypsinogen, chymotrypsinogen, and procarboxypeptidases
A and B, the zymogens of trypsin, chymotrypsin, and carboxypeptidases A and B,
are synthesized and secreted by the exocrine cells of the pancreas (Fig. 14–3b).
Trypsinogen is converted to its active form,
trypsin, by enteropeptidase, a proteolytic enzyme secreted by intestinal
cells. Free trypsin then catalyzes the conversion of additional trypsinogen to
trypsin. Trypsin also activates chymotrypsinogen, the procarboxypeptidases, and
proelastase.
Why this elaborate mechanism for getting active digestive enzymes into the
gastrointestinal tract? Synthesis of the enzymes as inactive precursors protects
the exocrine cells from destructive proteolytic attack. The pancreas further
protects itself against self-digestion by making a specific inhibitor, a protein
called pancreatic trypsin inhibitor, that effectively prevents premature
production of active proteolytic enzymes within the pancreatic cells.
Degradation of the short peptides in the small intestine is then completed by
other intestinal peptidases. These include carboxypeptidases A and B (both of
which are zinc-containing enzymes), which remove successive carboxyl-terminal
residues from peptides, and an aminopeptidase that hydrolyzes successive
amino-terminal residues from short peptides. The resulting mixture of free amino
acids is transported into the epithelial cells lining the small intestine (Fig.
18–3c), through which the amino acids enter the blood capillaries in the villi
and travel to the liver.
FIGURE 14–3 Part of the human digestive (gastrointestinal) tract. (a) The parietal cells and chief cells of the gastric glands secrete their products in response to the hormone gastrin. Pepsin begins the process of protein degradation in the stomach. (b) The cytoplasm of exocrine cells is completely filled with rough endoplasmic reticulum, the site of synthesis of the zymogens of many digestive enzymes. The zymogens are concentrated in membrane-enclosed transport particles called zymogen granules. When an exocrine cell is stimulated, its plasma membrane fuses with the zymogen granule membrane and zymogens are released into the lumen of the collecting duct by exocytosis. The collecting ducts ultimately lead to the pancreatic duct and thence to the small intestine. (c) Amino acids are absorbed through the epithelial cell layer (intestinal mucosa) of the villi and enter the capillaries. Recall that the products of lipid hydrolysis in the small intestine enter the lymphatic system after their absorption by the intestinal mucosa.
The first step in the catabolism of most L-amino acids, once they have reached the liver, is removal of the α- amino groups, promoted by enzymes called aminotransferases or transaminases. In these transamination reactions, the α-amino group is transferred to the α-carbon atom of α-ketoglutarate, leaving behind the corresponding α-keto acid analog of the amino acid. There is no net deamination (loss of amino groups) in these reactions, because the α-ketoglutarate becomes aminated as the α-amino acid is deaminated. The effect of transamination reactions is to collect the amino groups from many different amino acids in the form of L-glutamate. The glutamate then functions as an amino group donor for biosynthetic pathways or for elimination pathways that lead to the removal of nitrogenous waste products (Fig. 14-4).
FIGURE 14–4 Enzyme-catalyzed transaminations. In many aminotransferase reactions, α-ketoglutarate is the amino group acceptor. All aminotransferases have pyridoxal phosphate (PLP) as cofactor. Although the reaction is shown here in the direction of transfer of the amino group to α-ketoglutarate, it is readily reversible.
The enzymes are named for the amino group donor (alanine aminotransferase,
aspartate aminotransferase, for example). The reactions catalyzed by
aminotransferases are freely reversible, having an equilibrium constant of about
1.0 (ΔG'º = 0 kJ/mol).
All aminotransferases have the same prosthetic group and the same reaction
mechanism. The prosthetic group is pyridoxal phosphate (PLP), the
coenzyme form of pyridoxine, or vitamin B6. Pyridoxal phosphate functions as an
intermediate carrier of amino groups at the active site of aminotransferases. It
undergoes reversible transformations between its aldehyde form, pyridoxal
phosphate, which can accept an amino group, and its aminated form, pyridoxamine
phosphate, which can donate its amino group to an α-keto acid (Fig. 14–5a).
Pyridoxal phosphate is generally covalently bound to the enzyme’s active site
through an aldimine (Schiff base) linkage to the α-amino group of a Lys residue
(Fig. 14–5b, d).
FIGURE 14–5 Pyridoxal phosphate, the prosthetic group of aminotransferases. (a) Pyridoxal phosphate (PLP) and its aminated form, pyridoxamine phosphate, are the tightly bound coenzymes of aminotransferases. The functional groups are shaded. (b) Pyridoxal phosphate is bound to the enzyme through noncovalent interactions and a Schiffbase linkage to a Lys residue at the active site. (c) PLP (red) bound to one of the two active sites of the dimeric enzyme aspartate aminotransferase, a typical aminotransferase; (d) close-up view of the active site, with PLP (red, with yellow phosphorus) in aldimine linkage with the side chain of Lys258 (purple); (e) another close-up view of the active site, with PLP linked to the substrate analog 2-methylaspartate (green) via a Schiff base (PDB ID 1AJS).
Pyridoxal phosphate participates in a variety of reactions at the α, β, and γ carbons (C-2 to C-4) of amino acids. Reactions at the α carbon (Fig. 14–6) include racemizations (interconverting L- and D-amino acids) and decarboxylations, as well as transaminations. Pyridoxal phosphate plays the same chemical role in each of these reactions. A bond to the α carbon of the substrate is broken, removing either a proton or a carboxyl group. The electron pair left behind on the α carbon would form a highly unstable carbanion, but pyridoxal phosphate provides resonance stabilization of this intermediate (Fig. 14–6 inset). The highly conjugated structure of PLP (an electron sink) permits delocalization of the negative charge. Aminotransferases are classic examples of enzymes catalyzing bimolecular Ping-Pong reactions, in which the first substrate reacts and the product must leave the active site before the second substrate can bind. Thus the incoming amino acid binds to the active site, donates its amino group to pyridoxal phosphate, and departs in the form of an α-keto acid. The incoming α-keto acid then binds, accepts the amino group from pyridoxamine phosphate, and departs in the form of an amino acid.
FIGURE 14–6 Some amino acid transformations at the α carbon that are facilitated by pyridoxal phosphate. Pyridoxal phosphate is generally bonded to the enzyme through a Schiff base. Reactions begin (top left) with formation of a new Schiff base (aldimine) between the α-amino group of the amino acid and PLP, which substitutes for the enzyme-PLP linkage. Three alternative fates for this Schiff base are shown: (A) transamination (B) racemization, and C decarboxylation. The Schiff base formed between PLP and the amino acid is in conjugation with the pyridine ring, an electron sink that permits delocalization of an electron pair to avoid formation of an unstable carbanion on the α carbon (inset). A quinonoid intermediate is involved in all three types of reactions. The transamination route ( A ) is especially important in the pathways described in this chapter. The pathway highlighted here (shown left to right) represents only part of the overall reaction catalyzed by aminotransferases. To complete the process, a second α-keto acid replaces the one that is released, and this is converted to an amino acid in a reversal of the reaction steps (right to left). Pyridoxal phosphate is also involved in certain reactions at the β and γ carbons of some amino acids (not shown).
As we have seen, the amino groups from many of the α-amino acids are
collected in the liver in the form of the amino group of L-glutamate molecules.
These amino groups must next be removed from glutamate to prepare them for
excretion. In hepatocytes, glutamate is transported from the cytosol into
mitochondria, where it undergoes oxidative deamination catalyzed by
Lglutamate dehydrogenase (Mr 330,000). In mammals, this enzyme is present in
the mitochondrial matrix. It is the only enzyme that can use either NAD+ or
NADP+ as the acceptor of reducing equivalents. The combined action of an
aminotransferase and glutamate dehydrogenase is referred to as
transdeamination. A few amino acids bypass the transdeamination pathway and
undergo direct oxidative deamination. The α-ketoglutarate formed from glutamate
deamination can be used in the citric acid cycle and for glucose synthesis.
The free ammonia produced in tissues is combined with glutamate to yield
glutamine by the action of glutamine synthetase. This reaction requires
ATP and occurs in two steps (Fig. 14–7). First, glutamate and ATP react to form
ADP and a α-glutamyl phosphate intermediate, which then reacts with ammonia to
produce glutamine and inorganic phosphate. Glutamine is a nontoxic transport
form of ammonia; it is normally present in blood in much higher concentrations
than other amino acids. Glutamine also serves as a source of amino groups in a
variety of biosynthetic reactions. Glutamine synthetase is found in all
organisms, always playing a central metabolic role. In microorganisms, the
enzyme serves as an essential portal for the entry of fixed nitrogen into
biological systems. Glutamine in excess of that required for biosynthesis is
transported in the blood to the intestine, liver, and kidneys for processing. In
these tissues, the amide nitrogen is released as ammonium ion in the
mitochondria, where the enzyme glutaminase converts glutamine to glutamate and
NH4+. The NH4+ from intestine and kidney is transported in the blood to the
liver. In the liver, the ammonia from all sources is disposed of by urea
synthesis. Some of the glutamate produced in the glutaminase reaction may be
further processed in the liver by glutamate dehydrogenase, releasing more
ammonia and producing carbon skeletons for metabolic fuel.
FIGURE 14–7 Ammonia transport in the form of glutamine. Excess ammonia in tissues is added to glutamate to form glutamine, a process catalyzed by glutamine synthetase. After transport in the bloodstream, the glutamine enters the liver and NH4+ is liberated in mitochondria by the enzyme glutaminase.
Humans derive a small fraction of their oxidative energy from the catabolism of amino acids. Amino acids are derived from the normal breakdown (recycling) of cellular proteins, degradation of ingested proteins, and breakdown of body proteins in lieu of other fuel sources during starvation or in uncontrolled diabetes mellitus. Proteases degrade ingested proteins in the stomach and small intestine. Most proteases are initially synthesized as inactive zymogens. An early step in the catabolism of amino acids is the separation of the amino group from the carbon skeleton. In most cases, the amino group is transferred to α-ketoglutarate to form glutamate. This transamination reaction requires the coenzyme pyridoxal phosphate. Glutamate is transported to liver mitochondria, where glutamate dehydrogenase liberates the amino group as ammonium ion (NH4+). Ammonia formed in other tissues is transported to the liver as the amide nitrogen of glutamine or, in transport from skeletal muscle, as the amino group of alanine.
If not reused for the synthesis of new amino acids or
other nitrogenous products, amino groups are channeled
into a single excretory end product (Fig. 14–8). Most aquatic species, such as
the bony fishes, are ammonotelic, excreting amino nitrogen as ammonia. The toxic
ammonia is simply diluted in the surrounding water. Terrestrial animals require
pathways for nitrogen excretion that minimize toxicity and water loss. Most
terrestrial animals are ureotelic, excreting amino nitrogen in the form of urea;
birds and reptiles are uricotelic, excreting amino nitrogen as uric acid. Plants recycle virtually all amino groups, and
nitrogen excretion occurs only under very unusual
circumstances.
In ureotelic organisms, the ammonia deposited in
the mitochondria of hepatocytes is converted to urea in
the urea cycle. This pathway was discovered in 1932 by Hans Krebs (who
later also discovered the citric acid cycle) and a medical student associate,
Kurt Henseleit. Urea production occurs almost exclusively in the liver and is
the fate of most of the ammonia channeled there. The urea passes into the
bloodstream and thus to the kidneys and is excreted into the urine.
FIGURE 14–8 Urea cycle and reactions that feed
amino groups into the cycle. The enzymes catalyzing these reactions
(named in the text) are distributed between the mitochondrial matrix
and the cytosol. One amino group enters the urea cycle as carbamoyl
phosphate, formed in the matrix; the other enters as aspartate, formed
in the matrix by transamination of oxaloacetate and glutamate, catalyzed
by aspartate aminotransferase. The urea cycle consists of four
steps.
1 Formation of citrulline from ornithine and carbamoyl phosphate
(entry of the first amino group); the citrulline passes into the cytosol.
2 Formation of argininosuccinate through a citrullyl-AMP intermediate
(entry of the second amino group).
3 Formation of arginine
from argininosuccinate; this reaction releases fumarate, which enters
the citric acid cycle.
4 Formation of urea; this reaction also regenerates,
ornithine. The pathways by which NH4 arrives in the mitochondrial
matrix of hepatocytes were discussed in Section 18.1.
The urea cycle begins inside liver mitochondria, but
three of the subsequent steps take place in the cytosol;
the cycle thus spans two cellular compartments
(Fig. 14–8). The first amino group to enter the urea
cycle is derived from ammonia in the mitochondrial
matrix—NH4+ arising by the pathways described above.
The liver also receives some ammonia via the portal vein
from the intestine, from the bacterial oxidation of amino
acids. Whatever its source, the NH4+ generated in liver
mitochondria is immediately used, together with CO2
(as HCO3-) produced by mitochondrial respiration, to
form carbamoyl phosphate in the matrix (Fig. 18–11a;
see also Fig. 18–10). This ATP-dependent reaction is
catalyzed by carbamoyl phosphate synthetase I, a
regulatory enzyme. The mitochondrial form
of the enzyme is distinct from the cytosolic (II) form,
which has a separate function in pyrimidine biosynthesis.
The carbamoyl phosphate, which functions as an activated
carbamoyl group donor, now enters the urea cycle.
The cycle has four enzymatic steps:
Step 1) Carbamoyl phosphate donates its carbamoyl group to ornithine to form
citrulline, with the release of Pi. The reaction is catalyzed by ornithine
transcarbamoylase, and the citrulline passes from the
mitochondrion to the cytosol.
Step 2) The second amino group now enters from aspartate
(generated in mitochondria by transamination and transported
into the cytosol) by a condensation reaction
between the amino group of aspartate and the ureid
(carbonyl) group of citrulline, forming argininosuccinate.
This cytosolic reaction, catalyzed
by argininosuccinate synthetase, requires
ATP and proceeds through a citrullyl-AMP intermediate
(Fig. 14–9b).
Step 3) The argininosuccinate is cleaved by
argininosuccinase to form free
arginine and fumarate, the latter entering mitochondria
to join the pool of citric acid cycle intermediates. This
is the only reversible step in the urea cycle.
Step 4) In the last reaction of the urea cycle the cytosolic enzyme
arginase cleaves arginine to yield urea and ornithine.
Ornithine is transported into the mitochondrion
to initiate another round of the urea cycle.
FIGURE 14–9 Nitrogen-acquiring reactions in the synthesis of urea. The urea nitrogens are acquired in two reactions, each requiring ATP. (a) In the reaction catalyzed by carbamoyl phosphate synthetase I, the first nitrogen enters from ammonia. The terminal phosphate groups of two molecules of ATP are used to form one molecule of carbamoyl phosphate. In other words, this reaction has two activation steps (1 and 3). Carbamoyl Phosphate Synthetase I Mechanism (b) In the reaction catalyzed by argininosuccinate synthetase, the second nitrogen enters from aspartate. The ureido oxygen of citrulline is activated by the addition of AMP in step 1; this sets up the addition of aspartate in step 2, with AMP (including the ureido oxygen) as the leaving group.
Because the fumarate produced in the argininosuccinase reaction is also an intermediate of the citric acid cycle, the cycles are, in principle, interconnected—in a process named the “Krebs bicycle” (Fig. 14–10). However, each cycle can operate independently and communication between them depends on the transport of key intermediates between the mitochondrion and cytosol. Several enzymes of the citric acid cycle, including fumarase (fumarate hydratase) and malate dehydrogenase, are also present as isozymes in the cytosol. The fumarate generated in cytosolic arginine synthesis can therefore be converted to malate in the cytosol, and these intermediates can be further metabolized in the cytosol or transported into mitochondria for use in the citric acid cycle. Aspartate formed in mitochondria by transamination between oxaloacetate and glutamate can be transported to the cytosol, where it serves as nitrogen donor in the urea cycle reaction catalyzed by argininosuccinate synthetase. These reactions, making up the aspartate-argininosuccinate shunt, provide metabolic links between the separate pathways by which the amino groups and carbon skeletons of amino acids are processed.
FIGURE 14–10 Links between the urea cycle and citric acid cycle. The interconnected cycles have been called the “Krebs bicycle.” The pathways linking the citric acid and urea cycles are called the aspartate-argininosuccinate shunt; these effectively link the fates of the amino groups and the carbon skeletons of amino acids. The interconnections are even more elaborate than the arrows suggest. For example, some citric acid cycle enzymes, such as fumarase and malate dehydrogenase, have both cytosolic and mitochondrial isozymes. Fumarate produced in the cytosol—whether by the urea cycle, purine biosynthesis, or other processes—can be converted to cytosolic malate, which is used in the cytosol or transported into mitochondria to enter the citric acid cycle.
The flux of nitrogen through the urea cycle in an individual
animal varies with diet. When the dietary intake
is primarily protein, the carbon skeletons of amino acids
are used for fuel, producing much urea from the excess
amino groups. During prolonged starvation, when breakdown
of muscle protein begins to supply much of the
organism’s metabolic energy, urea production also increases
substantially.
These changes in demand for urea cycle activity are
met over the long term by regulation of the rates of synthesis
of the four urea cycle enzymes and carbamoyl
phosphate synthetase I in the liver. All five enzymes are
synthesized at higher rates in starving animals and in
animals on very-high-protein diets than in well-fed animals
eating primarily carbohydrates and fats. Animals
on protein-free diets produce lower levels of urea cycle
enzymes.
On a shorter time scale, allosteric regulation of at
least one key enzyme adjusts the flux through the urea
cycle. The first enzyme in the pathway, carbamoyl
phosphate synthetase I, is allosterically activated by
N-acetylglutamate, which is synthesized from
acetyl-CoA and glutamate by N-acetylglutamate synthase
(Fig. 14–11). In plants and microorganisms this enzyme
catalyzes the first step in the de novo synthesis of arginine
from glutamate, but in mammals
N-acetylglutamate synthase activity in the liver has a
purely regulatory function (mammals lack the other enzymes
needed to convert glutamate to arginine). The
steady-state levels of N-acetylglutamate are determined
by the concentrations of glutamate and acetyl-CoA (the
substrates for N-acetylglutamate synthase) and arginine
(an activator of N-acetylglutamate synthase, and thus
an activator of the urea cycle).
FIGURE 14–11 Synthesis of N-acetylglutamate and its activation of carbamoyl phosphate synthetase I.
If we consider the urea cycle in isolation, we see that
the synthesis of one molecule of urea requires four highenergy
phosphate groups (Fig. 14–8). Two ATP molecules
are required to make carbamoyl phosphate, and
one ATP to make argininosuccinate—the latter ATP undergoing
a pyrophosphate cleavage to AMP and PPi,
which is hydrolyzed to two Pi. The overall equation of
the urea cycle is
2NH4++ HCO3- + 3 ATP4- + H2O → urea + 2 ADP3-+ 4 Pi2- + AMP2- + 2 H+
However, the urea cycle also causes a net conversion of
oxaloacetate to fumarate (via aspartate), and the regeneration
of oxaloacetate (Fig. 14–10) produces NADH
in the malate dehydrogenase reaction. Each NADH molecule
can generate up to 2.5 ATP during mitochondrial
respiration, greatly reducing the overall
energetic cost of urea synthesis.
People with genetic defects in any enzyme involved in urea formation cannot tolerate proteinrich diets. Amino acids ingested in excess of the minimum daily requirements for protein synthesis are deaminated in the liver, producing free ammonia that cannot be converted to urea and exported into the bloodstream, and, as we have seen, ammonia is highly toxic. The absence of a urea cycle enzyme can result in hyperammonemia or in the build-up of one or more urea cycle intermediates, depending on the enzyme that is missing. Given that most urea cycle steps are irreversible, the absent enzyme activity can often be identified by determining which cycle intermediate is present in especially elevated concentration in the blood and/or urine. Although the breakdown of amino acids can have serious health consequences in individuals with urea cycle deficiencies, a protein-free diet is not a treatment option. Humans are incapable of synthesizing half of the 20 common amino acids, and these essential amino acids must be provided in the diet.
Ammonia is highly toxic to animal tissues. In the urea cycle, ornithine combines with ammonia, in the form of carbamoyl phosphate, to form citrulline. A second amino group is transferred to citrulline from aspartate to form arginine —the immediate precursor of urea. Arginase catalyzes hydrolysis of arginine to urea and ornithine; thus ornithine is regenerated in each turn of the cycle. The urea cycle results in a net conversion of oxaloacetate to fumarate, both of which are intermediates in the citric acid cycle. The two cycles are thus interconnected. The activity of the urea cycle is regulated at the level of enzyme synthesis and by allosteric regulation of the enzyme that catalyzes the formation of carbamoyl phosphate.
The pathways of amino acid catabolism, taken together, normally account for only 10% to 15% of the human body’s energy production; these pathways are not nearly as active as glycolysis and fatty acid oxidation. Flux through these catabolic routes also varies greatly, depending on the balance between requirements for biosynthetic processes and the availability of a particular amino acid. The 20 catabolic pathways converge to form only six major products, all of which enter the citric acid cycle (Fig. 14–12). From here the carbon skeletons are diverted to gluconeogenesis or ketogenesis or are completely oxidized to CO2 and H2O. All or part of the carbon skeletons of seven amino acids are ultimately broken down to acetyl-CoA. Five amino acids are converted to α-ketoglutarate, four to succinyl-CoA, two to fumarate, and two to oxaloacetate. Parts or all of six amino acids are converted to pyruvate, which can be converted to either acetyl-CoA or oxaloacetate. We later summarize the individual pathways for the 20 amino acids in flow diagrams, each leading to a specific point of entry into the citric acid cycle. In these diagrams the carbon atoms that enter the citric acid cycle are shown in color.
FIGURE 14–12 Summary of amino acid catabolism. Amino acids are grouped according to their major degradative end product. Some amino acids are listed more than once because different parts of their carbon skeletons are degraded to different end products. The figure shows the most important catabolic pathways in vertebrates, but there are minor variations among vertebrate species. Threonine, for instance, is degraded via at least two different pathways and the importance of a given pathway can vary with the organism and its metabolic conditions. The glucogenic and ketogenic amino acids are also delineated in the figure, by color shading. Notice that five of the amino acids are both glucogenic and ketogenic. The amino acids degraded to pyruvate are also potentially ketogenic. Only two amino acids, leucine and lysine, are exclusively ketogenic.
Seven amino acids (phenylalanine, tyrosine, isoleucine, leucine, tryptophan,
threonine, and lysine) are degraded entirely or in
part to acetoacetyl-CoA and/or acetyl-CoA.
These are the ketogenic amino acids (Fig. 14–12).
Their ability to form ketone bodies is particularly evident
in uncontrolled diabetes mellitus, in which the liver
produces large amounts of ketone bodies from both fatty
acids and the ketogenic amino acids.
The amino acids that are degraded to pyruvate, α-
ketoglutarate, succinyl-CoA, fumarate, and/or oxaloacetate
can be converted to glucose and glycogen. They are the
glucogenic amino acids. The division between ketogenic
and glucogenic amino acids is not sharp; five
amino acids (tryptophan, phenylalanine, tyrosine, threonine,
and isoleucine) are both ketogenic and glucogenic.
Catabolism of amino acids is particularly critical
to the survival of animals with high-protein diets or during
starvation. Leucine is an exclusively ketogenic amino
acid that is very common in proteins. Its degradation
makes a substantial contribution to ketosis under starvation
conditions.
A variety of interesting chemical rearrangements occur
in the catabolic pathways of amino acids. It is useful to
begin our study of these pathways by noting the classes
of reactions that recur and introducing their enzyme cofactors.
We have already considered one important
class: transamination reactions requiring pyridoxal
phosphate. Another common type of reaction in amino
acid catabolism is one-carbon transfers, which usually
involve one of three cofactors: biotin, tetrahydrofolate,
or S-adenosylmethionine. These cofactors
transfer one-carbon groups in different oxidation states:
biotin transfers carbon in its most oxidized state, CO2; tetrahydrofolate
transfers one-carbon groups in intermediate oxidation states and sometimes as
methyl groups; and S-adenosylmethionine transfers methyl groups, the most
reduced state of carbon. The latter two cofactors are especially important in
amino acid and nucleotide metabolism. Tetrahydrofolate (H4 folate), synthesized
in bacteria, consists of substituted pterin (6-methylpterin), p-aminobenzoate,
and glutamate moieties (Fig. 14–13).
FIGURE 14–13 Some enzyme cofactors important in one-carbon transfer reactions. The nitrogen atoms to which one-carbon groups are attached in tetrahydrofolate are shown in blue.
Most forms of tetrahydrofolate are interconvertible and serve as donors of one-carbon units in a variety of metabolic reactions. The primary source of one-carbon units for tetrahydrofolate is the carbon removed in the conversion of serine to glycine, producing N5,N10-methylenetetrahydrofolate. The oxidized form, folate, is a vitamin for mammals; it is converted in two steps to tetrahydrofolate by the enzyme dihydrofolate reductase. The one-carbon group undergoing transfer, in any of three oxidation states, is bonded to N-5 or N-10 or both. The most reduced form of the cofactor carries a methyl group, a more oxidized form carries a methylene group, and the most oxidized forms carry a methenyl, formyl, or formimino group (Fig. 14–14).
FIGURE 14–14 Conversions of one-carbon units on tetrahydrofolate. The different molecular species are grouped according to oxidation state, with the most reduced at the top and most oxidized at the bottom. All species within a single shaded box are at the same oxidation state. The conversion of N5,N10-methylenetetrahydrofolate to N5- methyltetrahydrofolate is effectively irreversible. The enzymatic transfer of formyl groups, as in purine synthesis and in the formation of formylmethionine in prokaryotes, generally uses N10-formyltetrahydrofolate rather than N5-formyltetrahydrofolate. The latter species is significantly more stable and therefore a weaker donor of formyl groups. N5-formyltetrahydrofolate is a minor byproduct of the cyclohydrolase reaction, and can also form spontancously. Conversion of N5-formyltetrahydrofolate to N5, N10-methenyltetrahydrofolate, requires ATP, because of an otherwise unfavorable equilibrium.
Although tetrahydrofolate can carry a methyl group
at N-5, the transfer potential of this methyl group is insufficient
for most biosynthetic reactions. S-Adenosylmethionine
(adoMet) is the preferred cofactor for biological
methyl group transfers. It is synthesized from
ATP and methionine by the action of methionine adenosyl
transferase (Fig. 14–15, step 1). This reaction
is unusual in that the nucleophilic sulfur atom of
methionine attacks the 5' carbon of the ribose moiety
of ATP rather than one of the phosphorus atoms. Triphosphate
is released and is cleaved to Pi and PPi on
the enzyme, and the PPi is cleaved by inorganic pyrophosphatase;
thus three bonds, including two bonds of
high-energy phosphate groups, are broken in this reaction.
The only other known reaction in which triphosphate
is displaced from ATP occurs in the synthesis of
coenzyme B12.
S-Adenosylmethionine is a potent alkylating agent
by virtue of its destabilizing sulfonium ion. The methyl
group is subject to attack by nucleophiles and is about
1,000 times more reactive than the methyl group of
N5-methyltetrahydrofolate.
Transfer of the methyl group from S-adenosylmethionine
to an acceptor yields S-adenosylhomocysteine
(Fig. 18–15, step 2), which is subsequently broken down
to homocysteine and adenosine (step 3). Methionine
is regenerated by transfer of a methyl group to homocysteine
in a reaction catalyzed by methionine synthase
(step 4), and methionine is reconverted to S-adenosylmethionine
to complete an activated-methyl cycle.
FIGURE 14–15 Synthesis of methionine and S-adenosylmethionine in an activated-methyl cycle. The steps are described in the text. In the methionine synthase reaction (step 4), the methyl group is transferred to cobalamin to form methylcobalamin, which in turn is the methyl donor in the formation of methionine. S-Adenosylmethionine, which has a positively charged sulfur (and is thus a sulfonium ion), is a powerful methylating agent in a number of biosynthetic reactions. The methyl group acceptor (step 2) is designated R.
The carbon skeletons of six amino acids are converted in whole or in part to pyruvate. The pyruvate can then be converted to either acetyl-CoA (a ketone body precursor) or oxaloacetate (a precursor for gluconeogenesis). Thus amino acids catabolized to pyruvate are both ketogenic and glucogenic. The six are alanine, tryptophan, cysteine, serine, glycine, and threonine (Fig. 14–16). Alanine yields pyruvate directly on transamination with α-ketoglutarate, and the side chain of tryptophan is cleaved to yield alanine and thus pyruvate. Cysteine is converted to pyruvate in two steps; one removes the sulfur atom, the other is a transamination. Serine is converted to pyruvate by serine dehydratase. Both the β-hydroxyl and the α-amino groups of serine are removed in this single pyridoxal phosphate–dependent reaction. Glycine is degraded via three pathways, only one of which leads to pyruvate. There are two significant pathways for threonine degradation. One pathway leads to pyruvate via glycine (Fig. 14–16). The conversion to glycine occurs in two steps, with threonine first converted to 2-amino-3- ketobutyrate by the action of threonine dehydrogenase. This is a relatively minor pathway in humans, accounting for 10% to 30% of threonine catabolism, but is more important in some other mammals.
FIGURE 14–16
Catabolic pathways for alanine, glycine,
serine, cysteine, tryptophan, and threonine.
Seven amino acids:
tryptophan, lysine, phenylalanine, tyrosine, leucine,
isoleucine, and threonine are converted into acetyl-CoA and/or
acetoacetyl-CoA, the latter being further converted to acetyl-CoA
(Fig. 14–17). Some of the final steps in the degradative
pathways for leucine, lysine, and tryptophan resemble
steps in the oxidation of fatty acids. Threonine
(not shown in Fig. 14–17) yields some acetyl-CoA via
the minor pathway illustrated in Figure 14–16.
The degradative pathways of two of these seven
amino acids deserve special mention. Tryptophan breakdown
is the most complex of all the pathways of amino acid
catabolism in animal tissues; portions of tryptophan
(four of its carbons) yield acetyl-CoA via acetoacetyl-
CoA. Some of the intermediates in tryptophan catabolism
are precursors for the synthesis of other biomolecules
(Fig. 14–18).
The breakdown of phenylalanine is noteworthy because
genetic defects in the enzymes of this pathway
lead to several inheritable human diseases. Phenylalanine and its oxidation
product tyrosine (both with nine carbons) are degraded
into two fragments, both of which can enter the citric
acid cycle: four of the nine carbon atoms yield free acetoacetate,
which is converted to acetoacetyl-CoA and
thus acetyl-CoA, and a second four-carbon fragment is
recovered as fumarate. Eight of the nine carbons of
these two amino acids thus enter the citric acid cycle;
the remaining carbon is lost as CO2. Phenylalanine, after
its hydroxylation to tyrosine, is also the precursor
of dopamine, a neurotransmitter, and of norepinephrine
and epinephrine, hormones secreted by the adrenal
medulla. Melanin, the black pigment of
skin and hair, is also derived from tyrosine.
FIGURE 14–17 Catabolic pathways for tryptophan, lysine, phenylalanine, tyrosine, leucine, and isoleucine. These amino acids donate some of their carbons (red) to acetyl-CoA. Tryptophan, phenylalanine, tyrosine, and isoleucine also contribute carbons (blue) to pyruvate or citric acid cycle intermediates. The fate of nitrogen atoms is not traced in this scheme; in most cases they are transferred to α- ketoglutarate to form glutamate.
FIGURE 14–18 Tryptophan as precursor. The aromatic rings of tryptophan give rise to nicotinate, indoleacetate, and serotonin. Colored atoms trace the source of the ring atoms in nicotinate.
The carbon skeletons of five amino acids (proline, glutamate,
glutamine, arginine, and histidine) enter the citric
acid cycle as α-ketoglutarate (Fig. 14–19). Proline,
glutamate, and glutamine have five-carbon skeletons.
The cyclic structure of proline is opened by oxidation of
the carbon most distant from the carboxyl group to
create a Schiff base, then hydrolysis of the Schiff base
to a linear semialdehyde, glutamate γ-semialdehyde.
This intermediate is further oxidized at the same carbon
to produce glutamate. The action of glutaminase,
or any of several enzyme reactions in which glutamine
donates its amide nitrogen to an acceptor,
converts glutamine to glutamate.
Transamination or deamination of
glutamate produces α-ketoglutarate.
Arginine and histidine contain five adjacent carbons
and a sixth carbon attached through a nitrogen
atom. The catabolic conversion of these amino acids to
glutamate is therefore slightly more complex than the
path from proline or glutamine. Arginine is
converted to the five-carbon skeleton of ornithine in the
urea cycle, and the ornithine is transaminated
to glutamate γ-semialdehyde. Conversion of histidine
to the five-carbon glutamate occurs in a multistep
pathway; the extra carbon is removed in a step that uses
tetrahydrofolate as cofactor.
FIGURE 14–19 Catabolic pathways for arginine, histidine, glutamate, glutamine, and proline. These amino acids are converted to α-ketoglutarate. The numbered steps in the histidine pathway are catalyzed by (1) histidine ammonia lyase, (2) urocanate hydratase, (3) imidazolonepropionase, and (4) glutamate formimino transferase.
The carbon skeletons of methionine, isoleucine, threonine, and valine are degraded by pathways that yield succinyl- CoA (Fig. 14–20), an intermediate of the citric acid cycle. Methionine donates its methyl group to one of several possible acceptors through S-adenosylmethionine and three of its four remaining carbon atoms are converted to the propionate of propionyl-CoA, a precursor of succinyl-CoA. Isoleucine undergoes transamination, followed by oxidative decarboxylation of the resulting α-keto acid. The remaining five-carbon skeleton is further oxidized to acetyl-CoA and propionyl-CoA. Valine undergoes transamination and decarboxylation, then a series of oxidation reactions that convert the remaining four carbons to propionyl-CoA. Some parts of the valine and isoleucine degradative pathways closely parallel steps in fatty acid degradation. In human tissues, threonine is also converted in two steps to propionyl-CoA. This is the primary pathway for threonine degradation in humans. The mechanism of the first step is analogous to that catalyzed by serine dehydratase, and the serine and threonine dehydratases may actually be the same enzyme.
FIGURE 14–20 Catabolic pathways for
methionine, isoleucine, threonine, and valine.
After removal of their amino groups, the carbon skeletons of amino acids undergo oxidation to compounds that can enter the citric acid cycle for oxidation to CO2 and H2O. The reactions of these pathways require a number of cofactors, including tetrahydrofolate and S-adenosylmethionine in one-carbon transfer reactions and tetrahydrobiopterin in the oxidation of phenylalanine by phenylalanine hydroxylase. Depending on their degradative end product, some amino acids can be converted to ketone bodies, some to glucose, and some to both. Thus amino acid degradation is integrated into intermediary metabolism and can be critical to survival under conditions in which amino acids are a significant source of metabolic energy. The carbon skeletons of amino acids enter the citric acid cycle through five intermediates: acetyl-CoA, α-ketoglutarate, succinyl-CoA, fumarate, and oxaloacetate. Some are also degraded to pyruvate, which can be converted to either acetyl-CoA or oxaloacetate. The amino acids producing pyruvate are alanine, cysteine, glycine, serine, threonine, and tryptophan. Leucine, lysine, phenylalanine, and tryptophan yield acetyl-CoA via acetoacetyl-CoA. Isoleucine, leucine, threonine, and tryptophan also form acetyl-CoA directly. Arginine, glutamate, glutamine, histidine, and proline produce α-ketoglutarate; isoleucine, methionine, threonine, and valine produce succinyl-CoA; four carbon atoms of phenylalanine and tyrosine give rise to fumarate; and asparagine and aspartate produce oxaloacetate.
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