The Nitrogen Economy and Protein Turnover

Metabolic Consequences of the Absence of Nitrogen Storage Compounds

Schoenheimer’s group synthesized 15N-labeled tyrosine and discovered that following administration of this tracer to
a rat, only about 50% of the 15N was recovered in the urine. Most of the remainder was incorporated into tissue
proteins. Importantly, only a small amount of the 15N found in the tissue proteins was attached to the original
tyrosine carbon skeleton—most had been incorporated into other amino acids. Finally, they observed that an
equivalent amount of unlabeled protein nitrogen was excreted, keeping the rat in nitrogen equilibrium.+

Protein Turnover
Proteins are subject to continuous biosynthesis and degradation, a process called protein turnover. As revealed in
Schoenheimer’s 15N experiments, many of the amino acids released during protein turnover are reutilized in the
synthesis of new proteins.

Proteolysis requires an input of energy; this is a surprising finding given that amide bond hydrolysis is exergonic. This
energy requirement reflects the fact that the process is nonrandom and highly regulated. Indeed, protein molecules
that have become chemically altered are preferentially degraded. A certain chemical change may mark a protein
molecule, targeting it for degradation by a proteolytic enzyme that specifically recognizes the marker.
The Nitrogen Economy and Protein Turnover
Intracellular Proteases and Sites of Turnover

Eukaryotes possess several types of intracellular proteases. Cathepsins

are contained in lysosomes, which form by budding from the Golgi
complex.
In addition to cathepsins, eukaryotic cells also possess many
nonlysosomal
proteases, including a large (2.5 megadalton) multisubunit ATP-
dependent protease called the proteasome

FIGURE Structure of the human proteasome.

The proteasome consists of a 28-subunit core particle (also known as the
20S particle) capped on one or both ends by a 19-subunit regulatory
particle (also known as the 19S regulatory particle). The proteolytic active
sites are located within the large internal space (∼100 * 60 A) of the 20S
core particle. The 19S regulatory particle controls substrate entry into the
20S core particle. Proteins tagged with the protein ubiquitin pass through
the tube in an ATP-dependent fashion. This structure was generated by
averaging multiple cryo-electron micrographs of the human 26S
proteasome. Image produced from PDB ID: 5t0c
The Nitrogen Economy and Protein Turnover
Chemical Signals for Turnover—Ubiquitination

We still have much to learn about the signals that target proteins
for degradation, but it is clear that specific structural features
and sequences on proteins convey information about the
metabolic stability of the proteins. The best understood of these
structural features is ubiquitination.

Ubiquitin is a small (76-residue) protein expressed in all

eukaryotic cells—it derives its name from its widespread
(ubiquitous) distribution. Ubiquitin is covalently conjugated to
specific cellular proteins in an ATP-dependent reaction, which
condenses the C-terminal carboxyl group of ubiquitin with
specific lysine amino groups on target proteins, forming an
isopeptide bond.

The selection of target proteins is determined by a large family of

ubiquitin–protein ligases (E3 ligases). The human genome
encodes ~600 of these ligases.
The Nitrogen Economy and Protein Turnover
Coenzymes Involved in Nitrogen Metabolism

Read and resume (pp 584-590)

AMINO ACID DEGRADATION AND METABOLISM OF NITROGENOUS END PRODUCTS

In animals whose dietary protein intake exceeds the need for protein synthesis and other biosyntheses, the excess
protein is mostly degraded, with the carbon skeletons of the amino acids being metabolized in the citric acid cycle
and the amino acid nitrogen excreted as urea.
plants and bacteria generally can synthesize most of their own amino acids, and they regulate the anabolic pathways
so that excesses rarely develop.
Transamination Reactions
With a few exceptions, the first step in amino acid degradation involves removal of the a-amino group to give the
corresponding a-keto acid. This process is usually catalyzed by enzymes called transaminases or, more properly,
aminotransferases.
Aminotransferases use pyridoxal phosphate as cofactor, and the chemistry of transamination is shown in Figure 18.7.

Transamination reactions have equilibrium constants close to unity. Therefore, the direction in which a particular
transamination proceeds is controlled in large part by the intracellular concentrations of substrates and products.
This means that transamination can be used not only for amino acid synthesis but also for degradation of amino acids
that accumulate in excess of need. In degradation, the aminotransferase works in concert with glutamate
dehydrogenase, as exemplified by the degradation of alanine:
AMINO ACID DEGRADATION AND METABOLISM OF NITROGENOUS END PRODUCTS

CONCEPTS:
Amino acid degradation usually begins with conversion to the corresponding a-keto acid by transamination or
oxidative deamination.

Transamination is the reversible transfer of an amino group from an a-amino acid to an a-keto acid, with pyridoxal
phosphate as a
coenzyme.

Serum glutamateoxaloacetate transaminase (SGOT) and serum glutamate-pyruvate transaminase (SGPT) are
important in the clinical diagnosis of human disease. Abundant in heart and in liver, these enzymes are released as
part of the cell injury that occurs in myocardial infarction, infectious hepatitis, or other damage to either organ. Assays
of these enzyme activities in blood serum can be used both in diagnosis and in monitoring the progress of a patient
during treatment.
AMINO ACID DEGRADATION AND METABOLISM OF NITROGENOUS END PRODUCTS

Transport of Ammonia to the Liver

FIGURE 18.10 Transport of ammonia to the liver for urea synthesis. The carrier is glutamine in most tissues, but it is
alanine in muscle.
AMINO ACID DEGRADATION AND METABOLISM OF NITROGENOUS END PRODUCTS

The Krebs–Henseleit Urea Cycle

Urea is synthesized almost exclusively in the liver and then transported to the kidneys for excretion. The pathway,
which is cyclic, was discovered by Hans Krebs and Kurt Henseleit in 1932, five years before the other cycle for which
Krebs is famous.
When arginine was added, urea was produced in 30-fold molar excess over the amount of arginine administered.
Similar results were seen if either of two structurally related amino acids, ornithine or citrulline, was substituted for
arginine. Because these three amino acids seemed to function catalytically to promote urea synthesis, Krebs and
Henseleit proposed the existence of a cyclic pathway.
 AMINO ACID DEGRADATION AND METABOLISM OF NITROGENOUS
END PRODUCTS
The Krebs–Henseleit Urea Cycle

The source of the carbon and one nitrogen atom in urea is

carbamoyl phosphate, synthesized from NH4 + and HCO3 -
(reaction❶) by carbamoyl phosphate synthetase I (CPS I).
Carbamoyl phosphate reacts with ornithine, via the enzyme
ornithine transcarbamoylase, to give citrulline (reaction ❷).
The second nitrogen comes from aspartate, which reacts with
citrulline to form argininosuccinate, through the action of
argininosuccinate synthetase (reaction ❸). Next,
argininosuccinase cleaves argininosuccinate in a b-elimination
reaction to give arginine and fumarate (reaction ❹). Arginine is
cleaved hydrolytically by arginase, to regenerate ornithine and
yield one molecule of urea (reaction ❻).

Adult humans produce about 30 g of urea each day. This

expensive pathway consumes up to 50% of the ATP produced
in liver.

FIGURE The Krebs–Henseleit urea cycle.

PATHWAYS OF AMINO ACID DEGRADATION

Animals generate most of their metabolic energy from oxidation of carbohydrates and fats. Nevertheless, animals
obtain 10%–15% of their energy from the oxidative degradation of amino acids.

There is also the processes by which amino acids are deaminated and the amino groups are converted either to
ammonia or to the amino group of aspartate for the production of urea.

Each of the 20 amino acids has a unique carbon skeleton, each amino acid requires its own degradation pathway.
However, these 20 pathways all converge on just seven common metabolic intermediates:
• pyruvate,
• α-ketoglutarate,
• succinyl-CoA,
• fumarate,
• oxaloacetate,
• acetyl-CoA, and
• acetoacetate.
PATHWAYS OF AMINO ACID DEGRADATION

Amino acids whose carbon skeletons are degraded to one of these

five intermediates are referred to as glucogenic amino acids

Pyruvate Family of Glucogenic Amino Acids

FIGURE Fates of the amino acid carbon skeletons.

PATHWAYS OF AMINO ACID DEGRADATION

Oxaloacetate Family of Glucogenic Amino Acids

FIGURE Fates of the amino acid carbon skeletons.

PATHWAYS OF AMINO ACID DEGRADATION

Pyruvate Family of Glucogenic Amino Acids

Asparaginase is an interesting example of enzyme-based chemotherapy.

The E. coli enzyme is widely used in the treatment of childhood acute
lymphoblastic leukemia. Normal and malignant lymphocytes depend on
the uptake of asparagine from the blood for growth—-asparaginase
depletes circulating asparagine, and this leads to complete remission in
many cases.
 PATHWAYS OF AMINO ACID DEGRADATION

α-Ketoglutarate Family of Glucogenic Amino Acids

Histamine is produced from decarboxylation of histidine. A

large number of antihistamines, such as diphenylhydramine
and desloratadine, are used to treat allergies and other
inflammations.
Typically, these drugs prevent the binding of histamine to its FIGURE Fates of the amino acid carbon skeletons.
receptors.
PATHWAYS OF AMINO ACID DEGRADATION

Succinyl-CoA Family of Glucogenic Amino Acids

FIGURE Fates of the amino acid carbon skeletons.

PATHWAYS OF AMINO ACID DEGRADATION

Acetoacetate/Acetyl-CoA Family of Ketogenic Amino Acids

FIGURE Fates of the amino acid carbon skeletons.

PATHWAYS OF AMINO ACID DEGRADATION

Phenylalanine and Tyrosine Degradation

Phenylketonuria (PKU) is caused by hereditary

deficiency of phenylalanine hydroxylase. If left
untreated, PKU leads to profound mental
retardation.
FIGURE The phenylalanine hydroxylation system.
PATHWAYS OF AMINO ACID DEGRADATION

Phenylalanine and Tyrosine Degradation

The degradation of tyrosine to acetoacetate and fumarate

occurs in a complex multistep process that begins with its
transamination. The pathway involves two dioxygenases,
which incorporate both atoms from O2 into the product,
and ends with a cleavage reaction that gives acetoacetate
and fumarate.
AMINO ACID BIOSYNTHESIS

Biosynthetic Capacities of Organisms

• Many bacteria and most plants can synthesize all of their nitrogenous metabolites starting from a single nitrogen
source, such as ammonia or nitrate.
• Many microorganisms will use a preformed amino acid, when available, in preference to synthesizing that amino
acid.
• Preformed amino acids are required. For example, over the course of evolution, the genus Lactobacillus has lost
many biosynthetic capacities because they grow in milk, a very nutrient-rich environment. Some Lactobacillus
species must therefore be provided with most of the 20 amino acids to be grown in the laboratory.
• Mammals are intermediate, being able to biosynthesize about half of the amino acids in quantities needed for
growth and for maintenance of normal nitrogen balance.
The amino acids that must be provided in the diet to meet an animal’s metabolic needs are called essential amino acids
AMINO ACID BIOSYNTHESIS

Amino Acid Biosynthetic Pathways

AMINO ACID BIOSYNTHESIS

Amino Acid Biosynthetic Pathways

AMINO ACID BIOSYNTHESIS

Amino Acid Biosynthetic Pathways

FIGURE Metabolic interconversions and fates of serine and glycine.

AMINO ACID BIOSYNTHESIS

Amino Acid Biosynthetic Pathways

FIGURE Biosynthesis of isoleucine, leucine, and valine.

After the serine–threonine dehydratase reaction, one set of enzymes catalyzes the comparable reactions in valine and
isoleucine synthesis. In bacteria, each end product regulates its own synthesis by inhibiting a specific enzyme.
AMINO ACIDS AS BIOSYNTHETIC PRECURSORS

Beyond their incorporation into proteins, amino acids serve as precursors for a tremendous variety of other
important metabolites, such as:
• polyamines,
• glutathione,
• methyl groups,
• heme,
• neurotransmitters and
• other signaling molecules, and nucleotides.
Summary

• Although inorganic nitrogen is abundant, metabolism of most organisms is limited by nitrogen bioavailability.
Reduction of N2 in biological nitrogen fixation and reduction of nitrate in plant and bacterial metabolism generate
ammonia, which all organisms can utilize.
• Proteins are in a continual state of turnover and replacement. The turnover is partly for replacement of damaged
proteins and partly as the result of normal cellular regulatory mechanisms. Most amino acids released by protein
turnover are reutilized for protein synthesis.
• Transamination and numerous additional reactions undergone by amino acids use pyridoxal phosphate as a
coenzyme. Tetrahydrofolate binds one-carbon units at three different oxidation states, interconverts them, and
transfers them in the synthesis of purine nucleotides, thymidine nucleotides, and several amino acids. B12
coenzymes include methylcobalamin, which participates in methionine biosynthesis, and 5′-
deoxyadenosylcobalamin, the coenzyme for methylmalonyl-CoA mutase. Folate metabolism presents various
chemotherapeutic targets, and folate and B12 deficiencies both have important clinical consequences.
• When amino acids are degraded, either for catabolism of an oversupply or when needed for energy generation,
the first step is usually removal of the a-amino group, either through transamination or oxidative deamination. The
resultant ammonia is excreted directly (in fish), converted to uric acid (in most reptiles, insects, and birds), or
converted to urea (in mammals). Urea synthesis is a cyclic pathway involving ornithine and arginine as
intermediates.
Summary

• The capacity for amino acid synthesis varies greatly among organisms. Mammals require about half of the 20
common amino acids in the diet. Amino acids are synthesized from intermediates in the citric acid cycle, glycolysis,
and the pentose phosphate pathway.
• Amino acids play numerous roles as intermediates in the biosynthesis of other metabolites. Amino acids are
involved in the biosynthesis of purine nucleotides (glutamine, glycine, serine), pyrimidine nucleotides (aspartate,
glutamine), polyamines and methyl groups (methionine), glutathione (glutamate, cysteine, glycine), creatine
phosphate (arginine), neurotransmitters (tyrosine, tryptophan, glutamate, arginine), lignin, aromatic compounds
and pigments (phenylalanine), hormones (tyrosine, histidine), porphyrins (glycine and glutamate in plants), and
other amino acids. The roles of amino acids as neurotransmitters and neurotransmitter precursors are particularly
important, as are their roles in porphyrin synthesis.
The Diversity of Enzyme Function

An enormous number of different proteins act as enzymes. Many of these enzymes were given common names,
specially during the earlier years of enzymology. Some enzyme names, like triose phosphate isomerase, are
descriptive of the enzyme’s substrate and its function; others, like trypsin, are not. To reduce confusion, a rational
naming and numbering system has been devised. Enzymes are divided into six major classes, with subgroups and sub-
subgroups to define their functions more precisely. The major classes are as follows:
1. Oxidoreductases catalyze oxidation–reduction reactions.
2. Transferases catalyze transfer of functional groups from one molecule to another.
3. Hydrolases catalyze hydrolytic cleavage.
4. Lyases catalyze removal of a group from, or addition of a group to,
a double bond, or other cleavages involving electron rearrangement.
5. Isomerases catalyze intramolecular rearrangement.
6. Ligases catalyze reactions in which two molecules are joined.

Information for almost all currently known enzymes can be found in online atabases such as BRENDA
(BRaunschweig ENzyme DAtabase) or ExPASy (Expert Protein nalysis System).
First-Order Reactions

FIGURE 8.1 Determining the order and rate constant of an irreversible first-
order reaction. Graphs (a) and (b) analyze the rate of a single reaction, with
time expressed as multiples of the half-life (t1 > 2) of the reactant.
Note that for each interval of t1 > 2 the reactant concentration is halved.
Second-Order Reactions
Most biochemical processes cannot be described over their full course by equations as simple as Equation 8.6. One
reason is that many of the reactions and processes we encounter are reversible, and as product accumulates, the
reverse reaction becomes important. For example, we may have a reaction like the following:

Because A is being consumed in the reaction to the right and formed by the reaction to the left, the corresponding
rate equation is:

In the case where n and m both equal 1, k1 and k-1 are, respectively, the rate constants for the first-order forward and reverse
reactions. Such a reaction approaches a state of equilibrium, at which point the rates of the forward and reverse reactions
become equal, and so the observed rate becomes zero (i.e., there is no apparent change in [A] or [B] over time). Thus, at
equilibrium

A second-order reaction occurs typically when two molecules must come together to form products:
𝐴 + 𝐵→𝐶
This is illustrated by the binding of oxygen to myoglobin:
𝑘1
𝑀𝑏 + 𝑂2 ՜ 𝑘1 𝑀𝑏𝑂2

The rate for this process is given by:

Free energy diagram for a favorable reaction, drawn on the basis of thermodynamics alone, will look like FIGURE (a).
It shows the free energy of the system versus the reaction coordinate, a generalized measure of the progress of the
reaction through intermediate states. For a favorable reaction, the free energy of the products is lower than that
would already have reacted. This observation suggests that only a fraction of the molecules—those that are
sufficiently energetic—can undergo reaction.
Transition States and Reaction Rates

We now consider two strategies for increasing reaction rates:

(1) raising the temperature of the reaction, and
(2) lowering the free energy of the transition state. The standard free energy of activation, ΔG∙‡, represents the
additional free energy (above the average free energy of reactant molecules) that molecules must have to attain the
transition state.
If the activation barrier to the reaction is high, only a small fraction of the molecules will have enough energy to
surmount it, or only a small fraction of collisions will be energetic enough for the reaction to occur.