Amino Acid Metabolism (day-2)
What to Know
• What is the Metabolic Fate of Ammonium?
• How is Escherichia coli Glutamine Synthetase regulated?
• Understand general ways that organisms synthesize
amino acids
• Know the definition of essential versus nonessential
amino acids
• Understand the glutamate transaminase rxn
• Have a general understanding of the 3 key metabolic
pathways responsible for amino acid synthesis
• Understand the key features of amino acid degradation
with regard to the citric acid and urea pathways
• General understanding of classroom discussion of PKU,
albinism, parkinson’s and porphyrias
Fate of Ammonium Ions
• Only three major reactions introduce NH4
into cells:
• Glutamate dehydrogenase
• Glutamine synthetase
• Carbamoyl-phosphate synthetase I
(mitochondrial enzyme of urea cycle)
Glutamate Dehydrogenase
Glutamine Synthetase
(a) The glutamine
synthetase reaction. (b)
The reaction proceeds
by activation of the γcarboxyl group of Glu by
ATP, followed by
amidation by NH4+.
Carbamoyl-Phosphate Synthetase
• Reaction is:
NH4+ + HCO3- + 2ATP → H2N-COO-PO32- + 2ADP + Pi + 2H+
• This reaction is an early step in the urea cycle
• Note the name “synthetase”, which is reserved for
synthetic enzymes that use ATP
• Enzymes that synthesize but do not use ATP are
termed “synthases”
What Regulatory Mechanisms Act on Escherichia coli
Glutamine Synthetase?
• A Case Study in Regulation
• GS (a 600kD α12-dodecahexameric protein) in E. coli is
regulated in three ways:
– Feedback inhibition
– Covalent modification (interconverts between
inactive and active forms)
– Regulation of gene expression and protein
synthesis control the amount of GS in cells
• But no such regulation occurs in eukaryotic versions
of GS
The subunit organization of
bacterial glutamine synthetase.
(a) Schematic
(b) Molecular structure: note the
pairs of metal ions (dark blue)
that define the active sites.
Allosteric Regulation of Glutamine Synthetase
Glutamine Synthetase is Regulated by Covalent Modification
Covalent modification of GS: Adenylylation of Tyr-397 in the
glutamine synthetase polypeptide via an ATP-dependent reaction by
the converted enzyme adenylyl transferase.
The cyclic cascade system regulating the covalent
modification of GS.
Degree of adenylation, n, ~ GS activity so high [Gln] / [α-KG] ratio = cell nitrogen
sufficiency and GS becomes adenylated and inactivated.
How Do Organisms Synthesize Amino Acids?
• Plants and microorganisms can make all 20 amino acids and
all other needed N metabolites
• In these organisms, glutamate is the source of N, via
transamination (aminotransferase) reactions of α-keto acid
analogue of the amino acid
• Mammals can make only 10 of the 20 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 they are made from –
– i.e. Glu, Gln, Pro, Arg are all members of the αketoglutarate family because they are derived from citric
acid cycle intermediate α-ketoglutarate
Humans Synthesize Only 10 of the 20 Common Amino Acids
Essential vs Nonessential Amino Acids
Amino Acids Are Synthesized From a Limited
Number of Precursors
Major Amino Acid Pathways
• Carbon skeletons of all 20 amino acids
are derived from just seven metabolic
intermediates
• The seven intermediates are found in
three metabolic pathways:
1) 3 glycolytic pathway intermediates
2) 2 pentose phosphate pathway
intermediates
3) 2 citrate cycle intermediates
Note: Plants and bacteria are capable of
synthesizing all 20 of the amino acids
illustrated in the figure; yet amino
acid biosynthesis in animals is much
more restricted due to the lack of
many of the required enzymes.
Feedback inhibition plays a
pivotal role modulating amino
acid biosynthetic pathways
Amino Groups for Amino Acids Are Derived From
Glutamate in Transamination Rxns
• Amino Acid1 + α-Keto acid2
α-Keto acid1 + Amino acid2
Glutamate-dependent transamination of α-keto acid carbon skeletons
is a primary mechanism for amino acid synthesis.
Amino Groups for Amino Acids Are Derived From
Glutamate in Transamination Rxns
Glutamate-dependent transamination of α-keto acid carbon skeletons
is a primary mechanism for amino acid synthesis. The transamination
of oxaloacetate by glutamate to yield aspartate and α-ketoglutarate is
a prime example.
The Aspartate Family of Amino Acids Includes Asp,
Asn, Lys, Met, Thr, Ile
Aspartate biosynthesis via transamination of oxaloacetate by
glutamate.
The Aspartate Family of Amino Acids Includes Asp,
Asn, Lys, Met, Thr, Ile
Asparagine biosynthesis from Asp, Gln, and
ATP by asparagine synthetase. βAspartyladenylate is an enzyme-bond
intermediate.
Biosynthesis of three nonessential amino acids (alanine, aspartate and
asparagine) and six essential amino acids (methionine, threonine, lysine,
isoleucine, valine and isoleucine) in E. coli involves two interconnected
pathways utilizing pyruvate and oxaloacetate as precursors.
Asparagine and Leukemia
Metabolic degradation of the common amino acids
Glucogenic amino acids are shown in pink, ketogenic in blue
Shikimate Pathway
Aromatic amino acids are synthesized in
plants, fungi and bacteria by a pathway
involving formation of a hydrocarbon
rain following the condensation of
phosphoenolpyruvate and erythrose-4phosphate.
Chorismate is a precursor to the three
aromatic amino acids, tryptophan,
tyrosine and phenylalanine.
Herbicides target chorismate
“Roundup”
“Roundup Ready soybeans” via
gene gun insertion of DNA
Amino Acids are Precursors to other Biomolecules
• In 1945, Dr. David Sherman
discovered that glycine contributed
all for nitrogen’s to team with carbon
atoms coming from both glycine and
acetate.
Tyrosine
Precursor to several important molecules in metabolic
signaling and neurotransmission including epinephrine and
dopamine
Tyrosine is also the precursor to pigment molecules
called melanins that are produced from dopaquinone
Inborn Errors of Metabolism
• PKU occurs one in every 15,000 births
• Caused by the accumulation of phenylalanine in the blood (30-50
times higher than normal)
• The high phenylalanine level leads to production of metabolites such
as phenyl pyruvate, phenyl acetate and phenyllactate, all of
which are associated with neurological and developmental
problems
Phenylketonuriacs must be careful to avoid
processed foods and beverages containing
aspartame (aspartyl-phenylalanine methyl
ester)
James Schlatter
Phenylalanine hydroxylase gene located on
chromosome 12– autosomal recessive genetic
disease
Probability that two PKU carriers will have a
child with the disease is 25%
frequency of carriers equals 2%,
therefore, probability of a baby born with PKU
by random chance equals one in 10,000
Albinism is another autosomal
recessive disease
Defective gene = tyrosinase
tyrosinase deficiency
results in loss of hair and skin
pigments
Porphyrias
• Porphyrias are caused by either autosomal recessive or
autosomal dominant mutations.
• Inhibit porphyrin ring synthesis
Acute Intermittent Porphyria
• Defective gene = porphobilinogen deaminase with
dominant genetic phenotype
• Under normal conditions disease is often asymptomatic
but a variety of factors including hormones, drugs
or dietary changes can trigger stomach pain and
neurological problems
• More rare form = congenital erythropoietic
porphyria which causes defective gene
encoding uroporphyrinogen-III cosynthase
• Leads to several defective metabolites including
uroporphyrinogen-1which builds up in the
teeth causing them to turn reddish brown
and fluoresce under UV light
The combination of symptoms of congenital erythropoietic porphyria,
combined with common medieval practice of drinking animal blood as
a treatment for human ailments may have accounted for the legend of
vampires.