Amino Acid Metabolism

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Amino Acid Metabolism
Why is amino acid metabolism important? Amino
acids are the monomers from which proteins are
synthesized metabolites that can be consumed
for energy production precursors of
biologically active N compounds heme biologi
cally active amines glutathione nucleotides
(and nucleotide coenzymes) Amino acids occur in
two classes essential amino acids must be
obtained from the diet phenylalanine,
tryptophan, isoleucine, valine,
threonine, methionine, leucine, and lysine
non-essential amino acids may be
synthesized biologically
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The Body Does Not Maintain a Pool of Excess Amino
Acids Excess amino acids are converted to
metabolic intermediates pyruvate oxaloacetate Ace
tyl-CoA a-ketoglutarate
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Amino Acid Breakdown
Deamination ? ammonia ? aspartate NH2
group Ammonia and aspartate NH2 group ? urea ?
excretion Carbon skeletons (i.e.
a-keto-acids) converted to metabolic
intermediates
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Deamination Reactions Are Carried Out by
Aminotransferases (Transaminases)
amino acid a-ketoglutarate
a-keto acid glutamate
Example Alanine Aminotransferase
Alanine Aminotransferase
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Transamination Reactions Involve Interconversion
of a-ketoglutarate and glutamate oxaloacetate
and aspartate
amino acid a-ketoglutarate
a-keto acid glutamate
glutamate oxaloacetate
a-ketoglutarate aspartate
Transamination reactions involve no net
deamination
The amino groups of most amino acids are funneled
into glutamate or aspartate.
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Reversal of the Order of the Previous Steps
Transforms an a-keto acid into an Amino Acid
Transamination
Forward reaction removes NH2 from an amino
acid a-keto acid product. Reverse
reaction take a different a-keto acid turn it
into an amino acid
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Deamination of Glutamate Produces Ammonium
glutamate NAD(P) H2O
a-ketoglutarate NH4 NAD(P)H
Direct hydride transfer of Ha to the oxidant
leads to an imine intermediate. Subsequent
hydrolysis leads to a-ketoglutarate.
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Glutamate Dehydrogenase Is Allosterically
Regulated Inhibitors GTP and NADH Activators
ADP, leucine, and NAD
GTP Favours
ADP Favours GTP site distorted
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The Urea Cycle Converts Ammonia Into Urea (Less
Toxic)
ammonia
urea
uric acid
NH3
Ammonia is excreted by many marine
animals toxicity is limited because of dilution
in the aqueous environment. Urea is excreted by
most terrestrial vertebrates. Uric acid is
excreted by birds and reptiles. Uric acid is
associated with gout.
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Reactions of the Urea Cycle
Carbamoyl phosphate synthetase condensation of
NH3 and HCO3- and 2 ATP yields carbamoyl
phosphate Ornithine transcarbamoylase carbamoyl
phosphate ornithine yields citrulline Arginin
osuccinate synthetase citrulline aspartate
ATP yields argininosuccinate PPi
AMP Argininosuccinase argininosuccinate
reactant yields fumarate and arginine Arginase
arginine reactant yields urea and ornithine
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Carbamoyl Phosphate Synthetase
HCO3- activated by phosphorylation source of urea
carbonyl carbon formation of carboxyphosphate cons
umption of ATP First of two urea nitrogen atoms.
Carboxyphosphate captured by ammonia elimination
of phosphate formation of carbamate
Carbamate trapped, stabilized by
phosphorylation yields carbamoyl
phosphate consumption of ATP
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Structure of E. coli Carbamoyl Phosphate
Synthetase
Glutamine binding site (production of ammonia in
E. coli) Carboxyphosphate domain site of
carboxyphosphate formation Carbamoyl phosphate
domain site of carbamoyl phosphate
formation Oligomerization domain Allosteric
regulatory domain
Carbamoyl phosphate synthetase exhibits a tunnel.
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Significance of the Carbamoyl Phosphate
Synthetase Tunnel
Half life for degradation of carboxyphosphate (pH
7, water) 28 ms Half life for degradation of
carbamate (pH 7, water) 70 ms
Ammonia produced travels 45 Å to react with
carboxyphosphate. Carbamate produced travels 35
Å to react with ATP. The tunnel protects against
degradation. Hence the logic of three catalytic
sites in one protein. Channeling
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Ornithine Transcarbamoylase
ornithine (lysine less 1 methylene group)
citrulline
ornithine transcarbamoylase
carbamoyl phosphate
Ornithine and citrulline are non-standard amino
acids.
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Argininosuccinate Synthetase
Second urea nitrogen atom enters with
aspartate. Citrulline and aspartate are
condensed to form argininosuccinate. Occurrence
of a citrullyl-AMP intermediate inferred
from observation of labeled oxygen transfer to
AMP product.
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Argininosuccinase Converts Argininosuccinate into
Fumarate and Arginine
fumarate
argininosuccinase

arginine
Arginine is the immediate metabolic precursor of
urea.
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Arginase Hydrolyses Arginine to Ornithine and
Urea
ornithine
arginase

H2O
urea
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The urea cycle.
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Regulation of the Urea Cycle
Regulation occurs at carbamoylphosphate
synthetase. Carbamoylphosphate synthetase
activated by N-acetyl glutamate. As amino acid
breakdown ? Urea synthesis must ? glutamate
varies as amino acid breakdown rates As
glutamate ? N-acetylglutamate ? As
N-acetylglutamate ? Carbamoylphosphate synthetase
activity ?
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Breakdown of Amino Acid Carbon Skeletons
Amino acids are either glucogenic ? glucose
biosynthetic precursors ketogenic ? acetyl-CoA,
acetoacetate or both.
Ketogenic leucine lysine eg leucine ?
acetyl-CoA acetoacetate
Both isoleucine phenylalanine threonine tryptoph
an tyrosine
Glucogenic all remaining amino acids. A, C, D,
E, G, H, M, N, P, Q, R, S, V
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Ketogenic Amino Acids Are Degraded to Acetyl-CoA
or Acetoacetate
Acetoacetate can be converted to
acetyl-CoA (recall fatty acid metabolism)
Glucogenic Amino Acids are Degraded to Glucose
Precursors pyruvate a-ketoglutarate succinyl-CoA
fumarate oxaloacetate
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Alanine is glucogenic
glucose (via gluconeogenesis)
alanine
pyruvate
Leucine is ketogenic
leucine
acetyl-CoA acetoacetate
Isoleucine is both ketogenic and glucogenic
isoleucine
succinyl-CoA acetyl-CoA
glucose (via gluconeogenesis)
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A, C, G, S, T ? Pyruvate (I)
Threonine dehydrogenase
Serine hydroxymethyl- transferase
a-amino- b-ketobutyrate lyase
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A, C, G, S, T ? Pyruvate (II)
Serine hydroxymethyl- transferase
alanine aminotransferase
serine dehydratase
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More PLP Chemistry The Serine Dehydratase
Reaction
non-enzymatic tautomerization
PLP-dependent but proceeds through b-elimination
of water, rather than tautomerization/deamination.
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More PLP Chemistry Serine Hydroxymethyl
Transferase
serine hydroxymethyl transferase

In serine hydroxymethyl transferase chemistry the
Ca-Cb bond is broken to produce a
resonance- stabilized carbanion.
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How Does PLP Facilitate Cleavage of Different
Bonds at Ca?
For delocalization, a particular geometry is
required so that the p system and the carbanion
orbital overlap. Different PLP-enzymes place
different Ca substituents in this orientation.
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From the Crystal Structure of Aspartate Amino
Transferase
a-methyl aspartate (inhibitor) complex with
PLP. Note that the methyl group (replacing Ha) is
virtually perpendicular to the PLP plane
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N,D ? Oxaloacetate
Transaminase activity on aspartate directly
yields oxaloacetate
transaminase
aspartate
oxaloacetate
Asparagine must first be deaminated to aspartate,
then converted (as above) to oxaloacetate
asparaginase
H2O
NH3
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R, Q, H, P ? a-ketoglutarate (I)
urocanate hydratase
histidine ammonia lyase
proline oxidase
Arginase
ornithine- d-aminotransferase
imidazolone proprionase
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R, Q, H, P ? a-ketoglutarate (III)
glutamate formimino- transferase
glutamate-5-semialdehyde dehydrogenase
glutaminase
glutamate dehydrogenase
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Methionine Metabolism (I)
methionine adenosyltransferase
methionine synthase
adenosyl- homocysteinase
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adenosyl homocysteinase
cystathionine b-synthase
cystathione g-lyase
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Methionine Metabolism (IV)
a-keto acid dehydrogenase
proprionyl-CoA carboxylase methylmalonyl-CoA
racemase methylmalonyl-CoA mutase
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The Tryptophan Degradation Pathway (I)
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The Tryptophan Degradation Pathway (II)
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The Tryptophan Degradation Pathway (III)
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The Tryptophan Degradation Pathway (IV)

The lysine degradation pathway.
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Step 4 of Tryptophan Degradation is PLP
Dependent Cb Cg Bond Cleavage (I)
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Step 4 of Tryptophan Degradation is PLP
Dependent Cb Cg Bond Cleavage (II)
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Step 4 of Tryptophan Degradation is PLP
Dependent Cb Cg Bond Cleavage (III)
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Amino Acids as Biosynthetic Precursors The
Biosynthesis of Heme
Heme occurs in hemoglobin myoglobin cytochro
me c (other electron transport
proteins) other cytochromes (e.g., P450s
drug metabolism) related to chlorophyll
Heme
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Amino Acids as Biosynthetic Precursors The
Biosynthesis of Heme
Heme is derived from glycine and acetate
(succinate). All of the nitrogen atoms in
heme are derived from glycine.
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The Citric Acid Cycle
After a number of rounds of the TCA cycle, the a-
and b- carbons of succinate arise from the methyl
group of acetyl-CoA. The g- and d- carbons arise
from the most recently added acetyl-CoA.
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Heme Synthesis (I) The Condensation of Glycine
and Succinyl-CoA to Yield ALA
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The Condensation of Glycine and Succinyl-CoA to
Yield ALA (II)
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Heme Synthesis (I) The Condensation of Two ALA
to Yield PBG
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The Condensation of Two ALA to Yield PBG (II)
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Phorbobilinogen Deaminase Chemistry Activation
of the Pyrrole Ring for Addition
General base catalyzed elimination of NH3
generates a methylene pyrrolinene intermediate
A acetyl P propionyl
Pyrrole
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Phorbobilinogen Deaminase Chemistry Sequential
Condensation of Four PBG
Enzyme is conjugated to dipyrromethane site of
attachment of first PBG.
Alkene polymerization chemistry.
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Phorbobilinogen Deaminase Chemistry
Continued Hydrolysis of the Methylbilane Enzyme
Yields The Porphyrin Precursor
Hydroxymethyl bilane has two fates.
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Hydroxymethyl bilane is Converted into
Uroporphyrinogen I and III
Spontaneous
Enzymatic Uroporphyrinogen III Synthase
Uroporphyrinogen III (Asymmetric)
Uroporphyrinogen I (Symmetric)
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Conversion of Uroporphyrinogen III to Heme
The four acetyl groups A are decarboxylated to
methyl groups. The two propionyl P groups across
from the asymmetric center are decarboxylated to
vinyl groups. The methylene groups (R-CH2-R)
linking the pyrrole rings are oxidized to methine
(R-CHR) groups. An Fe2 is added and becomes
coordinated by the four N atoms, one from each
pyrrole ring.
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Synthesis of Physiologically Active Amines GABA
GABA g-amino-butyric acid
A major inhibitory neurotransmitter. Expressed at
30 of synapses.
GABA is derived from glutamic acid
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GABA is Formed From Glutamate in a One Step,
PLP-dependent Decarboxylation
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Histamine Is Derived from Histidine in a
PLP-dependent Decarboxylation
Histamine is known primarily as a mediator of the
inflammatory response in the immune system. It
is also a neurotransmitter. Recent evidence
suggests that it plays a role in the chemotaxis
of white blood cells (also probably immune
related).
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Serotonin is Derived from Tryptophan
serotonin 5-hydroxy tryptamine
Serotonin is a major neurotransmitter stimulates
smooth muscle contraction. Appears to play a
role in mood. Many antidepressants are Selective
Serotonin Reuptake Inhibitors (SSRIs)
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Biosynthesis of Serotonin
Hydroxylation at the 5- position of the indole
ring followed by PLP-dependent decarboxylation
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Tyrosine is the Precursor for a Variety of
Molecules
Addition of a hydroxyl at the meta position of
the tyrosine ring yields dihydroxyphenylalanine, L
-DOPA. L-DOPA is the precursor to the skin
pigment malanin and to dopamine.
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Dopamine is Derived From L-DOPA by Decarboxylation
Insufficient levels of dopamine result in the
tremors associated with Parkinsons
disease. Functions in motor activity, mood,
attention, sleep, learning, and the
reward system. Important in addiction research.
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Norepinephrine and Epinephrine (Adrenaline) Are
Derived from Dopamine
Mediators of the fight or flight
response. Increases blood flow to skeletal
muscle. Increased availability of oxygen,
increased availability of glucose (stimulates
glycogen catabolism).
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The following slides are supplemental.
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Review of Transamination Chemistry The
Pyridoxal-5-phosphate Electron Sink
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Transamination Reactions (I) The Enzyme-PLP
Schiff Base Is Exchanged for an Amino Acid-PLP
Schiff Base (Transimination)
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Transamination Reactions (II) The Aldimine
Intermediate is Deprotonated and Tautomerizes
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Transamination Reactions (III) Hydrolysis
Releases the a-keto acid Product and the
Enzyme-NH2 Product