Chapter 20

Metabolism of
Nitrogenous compounds I:
Principles of Biosynthesis,
Utilization, and Turnover
 Outline
 The Nitrogen Cycle
 Ammonium assimilation
 The Nitrogen Economy: Aspects of Amino Acid
Synthesis and Degradation
 Amino Acid Degradation and Metabolism of
Nitrogenous End Products
 Coenzymes Involved in Nitrogen Metabolism
 How Do the Various Organic Forms of
Nitrogen Arise?
 The prevalent forms of nitrogen in the environment
are inorganic and oxidized:
 N2 (dinitrogen gas)
 NO3- (nitrate anion)
 Nitrogen in organic compounds is predominantly in a
reduced state
 The two principal routes for organic nitrogen
acquisition
 Nitrate assimilation (NO3- to NH4+): 99%
 Nitrogen fixation (N2 to NH4+): 1%
 Both these lead to formation of ammonium ions
 Depends on green plants and microorganisms
 Reactions that incorporate ammonium ion into organic
molecules follow
The nitrogen cycle
Nitrogenase complex
Organic,
reduced

nitrite reductase

Inorganic, nitrate reductase

oxidized

Nitrogen fixation and nitrate reduction convert inorganic to

organic nitrogen
Denitrification and nitrogen fixation are anaerobic processes
N2 reduction
• Rhizobium bacteria fix nitrogen in symbiotic association
with leguminous plants
• N2 reduction is catalyzed by the enzyme nitrogenase
• 4 types
• The most abundant and widely studied is the molybdenum
(Mo)-dependent nitrogenase
• The stoichiometry of the overall reaction is as follows (6
e- for NH3 and 2 e- for H2 formation; 2ATP for 1 e- )

• Industrially, the reduction is done by the Haber–Bosch

process, a low-yield catalytic hydrogenation carried out
at high temperature and pressure
• This process is used in the manufacture of ammonia-
based fertilizers Copyrigh
t © 2013
Pearson
Canada
Inc.
 The two-component molybdenum-
dependent nitrogenase reaction

• Fe protein, dinitrogenase reductase,

component II
• The Fe protein undergo a three-state
cycle
• Binding of ATP to reduced Fe protein
• hydrolysis of bound ATP to oxidized Fe
protein
• Transfer of the electron to MoFe protein
• MoFe protein, dinitrogenase ,
component I
• a nine-state cycle
• catalyzes the reduction of N2 and is
reduced by Fe protein
 Molybdenum (Mo)-dependent
nitrogenase
• Fe protein:
• Homodimer, twoATP binding
sites
• A Fe4–S4 cluster
• MoFe protein:
• An 22-heterotetramer
• Contains
• a Fe7-S8 P cluster
• a iron–molybdenum cofactor
(FeMo-co, Fe9-S7-Mo ): the
molybdenum atom
coordinated to a molecule of
R-homocitrate through its
2-hydroxy and 2-carboxyl
groups
 Structure of molybdenum-dependent
nitrogenase from Azotobacter vinelandii
• Fe-protein: The two subunits of the
homodimeric Fe protein each containing a
bound MgADP and the bridging Fe4–S4
cluster
• FeMo protein: Each α unit binds one P
cluster and one FeMo-co cluster

• Reaction:
• Binding of ATP forces Fe protein to duck
to the MoFe protein
• Hydrolysis of bound ATP is thought both
• to drive the reduction of P cluster by
Fe protein
• to trigger a conformational change in
Fe protein and Fe protein dissociates
transiently from MoFe protein,
assuring unidirectional electron flow
Nitrate Utilization-nitrate reductase

• The ability to reduce nitrate to ammonia is

common to virtually all plants, fungi, and
bacteria
• The first step, reduction of nitrate (+5
oxidation state) to nitrite (+3 oxidation state)
is catalyzed by nitrate reductase
• The eukaryotic enzyme contains bound FAD,
molybdenum, and a cytochrome b5
• Electrons are transferred from NAD(P)H to
enyzyme-FAD, then to cytochrome b5 , then to
molybdenum, and to substrate
• The enzyme carries out the overall reaction:
 Nitrate Utilization-nitrate
reductase
• The molybdenum cofactor in
nitrate reductase is bound to a
cofactor containing a pteridine
ring, which is quite distinct from
the structure of FeMo-co

• In fact, all known molybdenum-

requiring enzymes except
nitrogenase contain a structure
similar to that of this
molybdopterin, in which the Mo
center shows a pyramidal geometry
Nitrite Utilization-Nitrite reductase

• Reduction of nitrite to ammonia is

carried out in three steps by one
enzyme, nitrite reductase

• Higher plants, algae, and cyanobacteria

use ferredoxin as the electron donor in
this six-electron reaction.

• This enzyme contains one Fe4S4 center

and one molecule of siroheme, a partially
reduced iron porphyrin
 Outline
 The Nitrogen Cycle
 Ammonium assimilation
 The Nitrogen Economy: Aspects of Amino Acid
Synthesis and Degradation
 Amino Acid Degradation and Metabolism of
Nitrogenous End Products
 Coenzymes Involved in Nitrogen Metabolism
Reactions in assimilation of
ammonia

1. Glutamate
dehydrogenase
2. Glutamine synthetase
3. Asparagine synthetase
4. Carbamoyl phosphate
synthetase
5. Glutamate synthase.

•In animals, glutamine,

rather than NH3, is the
chief nitrogen source for
pyrimidines
Glutamate dehydrogenase: Reductive
amination of -ketoglutarate
• A hexamer of identical subunits, located in
the inner mitochondrial membrane
• Is activated under conditions of low energy
charge
• Is reversible reaction, high KM for ammonia
(~1mM)
Glutamate synthase
• A reaction comparable to that catalyzed by
glutamate dehydrogenase but functions primarily
in glutamate biosynthesis in most cells where
ammonia levels are lower
• 2 subunits (α, β) in an 800-kDa holoenzyme
containing FMN, FAD, [4Fe-4S] clusters
• Plants use NADPH, NADH, or ferredoxin, other
organisms use NADH exclusively
Glutamine synthetase
• Glutamate can accept a second
ammonia moiety to form
glutamine in the reaction
catalyzed by
• Mn2+ is required
• ATP-dependent amidation of γ-
carboxyl of glutamate to
glutamine: acyl phosphate
intermediate
Glutamine synthetase
• Bacterial enzyme: a dodecamer of identical
subunits forming 2 facing hexagonal arrays
• Activity is tightly regulated
• A key participant in detoxifying ammonia in brain
• Accumulation of glutamate and glutamine can
deplete α-KG, interfering with citric acid cycle
Regulation of Glutamine synthetase
• Prokaryotic glutamine synthetase is controlled by:
o Allosteric regulation: Cumulative feedback inhibition
▪ Involves the action of eight specific feedback inhibitors
▪ Metabolic end products of glutamine (Trp, His,
glucosamine-6-phosphate, carbamoyl phosphate, CTP, and
AMP)
▪ Indicators of the general status of amino acid
metabolism (Ala, Gly)
o Covalent modification
▪ Adenylylation of a specific tyrosine residue (Tyr 397)
▪ Low Gln, PII-UMP controls GS gene expression
▪ Mammalian GS
▪ A homodecamer, composed of two pentameric rings
▪ is feedback inhibited by carbamoyl phosphate, Gln, several
other a.a.(liver); only by carbamoyl phosphate (brain)
▪ no covalent modification
 Regulation of the activity of
bacterial glutamine synthetase

• AT :adenylyltransferase
• GS: glutamine synthetase
• UT: uridylyltransferase
• UR: uridylyl-removing
enzyme
Two principal pathways of Ammonium
Assimilation Lead to Glutamine Synthesis
 When NH4+ is enough
 the glutamate dehydrogenase/glutamine synthetase
pathway

 When little NH4+ is available

 the glutamate synthase/glutamine synthetase pathway
Asparagine Synthetase

• Catalyzes a reaction comparable to that of

glutamine synthetase
• Although widespread, asparagine synthetase
accounts for much less ammonia assimilation
• Glutamine is strongly preferred as a
substrate over ammonia
Carbamoyl phosphate synthetase
• The final route for assimilating ammonia
first forms carbamoyl phosphate
• This reaction is an early step in the urea
cycle
• Either ammonia or glutamine can serve as
the nitrogen donor
• Eukaryotic cells have two forms:
• Form I: mitochondria, has a preference
for ammonia
• Form II: cytosol, has a preference for
glutamine, is inhibited by uridine
triphosphate
 Outline
 The Nitrogen Cycle
 Ammonium assimilation
 The Nitrogen Economy: Aspects of Amino Acid
Synthesis and Degradation
 Amino Acid Degradation and Metabolism of
Nitrogenous End Products
 Coenzymes Involved in Nitrogen Metabolism
Essential and Nonessential Amino Acids
 Plants and microorganisms can make all 20
amino acids (and all other needed N-
metabolites)
 In amino acid biosynthesis, glutamate is the
primary source of N, via transamination
(aminotransferase) reactions

 Mammals can make only 10 of the 20 amino

acids: non-essential amino acids
 The others are classed as "essential" amino
acids and must be obtained in the diet
 All amino acids are grouped into families
according to the intermediates that serve as
their precursors
Essential and Nonessential Amino
Acids
 Transamination

• 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.
 Transamination Reaction

• Aminotransferases utilize a
coenzyme, pyridoxal phosphate,
that is derived from vitamin B6
 Pyridoxal phosphate
• Pyridoxal phosphate is derived from
vitamin B6
• Vitamin B6 is also called pyridoxine
• Pyridoxal phosphate (PLP) is the
predominant coenzyme form
• The hydroxymethyl group at position
4 is oxidized to an aldehyde and at
position 5 is phosphorylated
• The aldehyde group forms aldimine,
reactive species with ε-amino acid of
Lys in the active site
• Pyridoxamine phosphate (PMP) is an
intermediate form in transamination
reactions
Transamination
Aspects of Amino Acid Synthesis and
Degradation
• Transamination can be used for amino acid
synthesis and also for degradation of amino acids
that accumulate in excess of need

• In degradation the transaminase works in concert

with glutamate dehydrogenase, as exemplified by
the degradation of alanine:
 Clinical diagnosis of human disease
• Most aminotransferases use glutamate/α-ketoglutarate as one
of the two α-amino/α-keto acid pairs involved
• Two such enzymes are important in the clinical diagnosis of
human disease—serum glutamate-oxaloacetate transaminase
(SGOT) and serum glutamate-pyruvate transaminase
(SGPT):

• These enzymes, abundant in heart and in liver, are released

from cells 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
 Outline
 The Nitrogen Cycle
 Ammonium assimilation
 The Nitrogen Economy: Aspects of Amino Acid
Synthesis and Degradation
 Amino Acid Degradation and Metabolism of
Nitrogenous End Products
 Coenzymes Involved in Nitrogen Metabolism
Protein Turnover
• Proteins are subject to continuous
biosynthesis and degradation, a process called
protein turnover

• For an intracellular protein whose total

concentration does not change with time, the
steady state level is maintained by synthesis
of the protein at a rate just sufficient to
replenish protein lost by degradation

• Many of the amino acids released during

protein turnover are reutilized in the
synthesis of new proteins
Protein Turnover
Structure of the proteasome
• The proteasome (26S) contains two major
assemblies, a 28-subunit core particle (the
20S particle) and a 19-subunit regulatory
particle (the 19S particle), which forms the
base and lid assemblies
• The proteolytic active sites are located
within the large internal space (~100 x 60Å)
of the 20S core particle
• The lid and base assemblies of the 19S
regulatory particle control substrate entry
into the 20S core particle
• Proteins tagged with the protein ubiquitin
pass through the tube in an ATP-dependent
fashion
Amino Acid Degradation and
Metabolism of Nitrogenous End
Products
• Amino acid degradation usually begin with
removal of a-amino group to give the
corresponding a-keto acid by transamination
(Aminotransferases) or oxidative deamination
(L-amino acid oxidase, a flavoprotein)
Amino Acid Degradation
 Glucogenic amino acids: whose carbon skeletons
generate intermediates for gluconeogenesis
 Pyruvate
 a-Ketoglutarate
 Succinyl-CoA
 Fumarate
 Oxaloacetate
 Ketogenic amino acids: whose carbon skeletons
generate intermediates for ketogenesis
 Acetyl-CoA
 Acetoacetate
 Some amino acids are both glucogenic and
ketogenic
* More than one route
Detoxification and excretion
of ammonia
• Ammonia participate in amino acid
synthesis and degradation
• Ammonotelic : release ammonia, e.g.
aquatic animals
• Ureotelic: release urea, e.g. Many
terrestrial vertebrates
• Uricotelic: release uric acid, e,g.
bird, reptiles, and insects
• The Krebs-Henseleit Urea cycle
• Was discovered by Hans Krebs
and Kurt Henseleit in 1932
• is synthesized almost exclusively
in the liver and then transported
to the kidneys for excretion
The Krebs-
Henseleit
urea cycle
The mechanism of action of CPS-1
Arginosuccinate synthetase mechanism
Arginine biosynthesis
 Argininebiosynthesis involves
enzymatic steps that are also part of
the urea cycle
 Requires ornithine biosynthesis from
glutamate
 Ornithine:
(1) a precursor to arginine; (2) an
intermediate in the urea cycle; (3) an intermediate in
Arg degradation
 Requires carbamoyl-phosphate from
ammonium assimilation
 Ornithine transcarbamoylase converts
ornithine to citrulline in the urea cycle
Ornithine biosynthesis from glutamate
N-acetylglutamate synthase

N-acetylglutamate kinase
N-acetylglutamate-5-
semialdehyde dehydrogenase

N-acetyl-ornithine-δ-aminotransferase

N-acetylornithine deacetylase
The urea cycle
• The net reaction for one turn of the urea
cycle is as follows:

• Urea is synthesized by an energy-requiring

cyclic pathway that begins and ends with
ornithine
• Only ureotelic organisms contains arginase, so
they can carry out the cyclic pathway
• Glutamate dehydrogenase, carbamoyl
phosphate synthetase I, ornithine
carbamoyltransferase are in mitochondria
Regulation of the urea cycle
• Long-term regulation
• Four urea cycle enzymes and CPS I are increased on high
protein diets, but decreased on protein-free diet
• Short-term regulation
• CPSI
• Allosteric activation by N-acetylglutamate (determined by
level of glutamate)
• Covalent modification:
• Acetylation of Lys inactivates enzyme
• Deacetylation by sirtuin (SIRT5,activated by mito
NAD+)
• Hyperammonemia (high blood NH4+ levels)
• Liver function test
• Blood urea nitrogen (BUN) levels
• Kidney function test
• Ammonia toxicity: ammonia deplete citric acid cycle
intermediates and NADH by overloading the glu dehydrogenase
reaction, leading ATP deficiency (brain is especially susceptible)
Transport of ammonia to the liver
• Most tissues convert ammonia to glutamine
but it is alanine in muscle
Transport of ammonia to the liver

• The glucose–alanine cycle removes toxic

ammonia from muscle

• Glutamine synthetase and glutaminase do the

same for most other tissues
 Outline
 The Nitrogen Cycle
 Ammonium assimilation
 The Nitrogen Economy: Aspects of Amino Acid
Synthesis and Degradation
 Amino Acid Degradation and Metabolism of
Nitrogenous End Products
 Coenzymes Involved in Nitrogen Metabolism
Coenzymes Involved in Nitrogen Metabolism

1. Pyridoxal phosphate, that is derived from

vitamin B6
• The cofactor for transamination reactions

2. The folic acid

• Transfer single-carbon functional group

3. The B12, cobalamin

• Participate in the synthesis of methionine
 Pyridoxal phosphate
• Pyridoxal phosphate is derived
from vitamin B6
• Vitamin B6 is also called
pyridoxine
• Pyridoxal phosphate (PLP) is
the predominant coenzyme
form
• The hydroxymethyl group at
position 4 is oxidized to an
aldehyde and at position 5 is
phosphorylated
• Pyridoxamine phosphate (PMP)
is an intermediate form in
transamination reactions
The Mechanism of the Aminotransferase
(Transamination) Reaction
Coenzymes Involved in Nitrogen Metabolism

1. Pyridoxal phosphate, that is derived from

vitamin B6
• The cofactor for transamination reactions

2. The folic acid

• Transfer single-carbon functional group

3. The B12, cobalamin

• Participate in the synthesis of methionine
Folic acid
 Folic acid, a B vitamin found in green plants,
fresh fruits, yeast, and liver, is named from
folium, Latin for “leaf”
 Mammals cannot synthesize folate
Conversion of folic acid to
Tetrahydrofolate

• Once inside a cell, folic acid is converted to

active forms by two successive reductions of
the pyrazine part of the pteridine ring

• Both reactions are catalyzed by the NADPH-

specific enzyme dihydrofolate reductase
 Tetrahydrofolate
• N-5, N-10 carry single carbon units
• A “tail” of glutamate: multiple glutamate
residues (3-8), which help them be retained
within cells and bind more tightly to enzymes
• Not peptide bond, γ-carboxyl group of the first with
α-amino group of the next
• Monoglutamated form is taken up efficiently in
animals
Tetrathydrofolate (THF)
N5-methyl-THF
• Folates are acceptors and donors
of one-carbon units for all
oxidation levels of carbon
• the methyl, methylene, and formyl
oxidation levels
Coenzymes Involved in Nitrogen Metabolism

1. Pyridoxal phosphate, that is derived from

vitamin B6
• The cofactor for transamination reactions

2. The folic acid

• Transfer single-carbon functional group

3. The B12, cobalamin

• Participate in the synthesis of methionine
 Structure of vitamin B12
• The molecule shown here is
the cyanide-containing form
originally isolated
(cyanocobalamin)
• In cells, a water molecule or
hydroxyl group takes the
place of CN, forming the
precursor to the coenzyme
forms of B12 (aquocobalamin,
hydroxocobalamin)
• The corrin ring is shown in red
• 5,6-Dimethylbenzimidazole
(DMB), which is linked to the
cobalt, is shown in blue
Coenzymes derived from vitamin B12

•The corrin ring, identical in all known

forms of B12
•The Co bears a positive charge (n = 1,
2, or 3), while each molecule is
uncharged overall
•B12 coenzymes have either a methyl
group or a 5’-deoxyadenosyl moiety
linked to cobalt, making them the first
known organometallics in metabolism
 The role of B12 as a methyl
transferase in Met biosynthesis

B12-dependent enzymes:
• isomerase
• Methyl-transferase
• uses methyl-B12
• Reductive dehalogenase
The intramolecular isomerization
catalyzed by methylmalonyl-CoA
mutase
B12 coenzymes and Pernicious anemia

• Pernicious anemia is caused by deficiency of a

glycoprotein (secreted by gastric tissues)
needed for intestinal absorption of vitamin B12,
leading to intracellular deficiencies of B12
coenzymes

• B12 deficiency causes 5-

methyltetrahydrofolate to accumulate, with
concomitant depletion of other folate
coenzymes

• Folic acid deficiency increases the risk of

cardiovascular disease and birth defects in
humans.
A relationship between folate and B12
metabolism
•This scheme is based on the apparent folate
deficiency seen in early stages of B12 deficiency
as a result of decreased flux through the
methionine synthase reaction