FCH 532 Lecture 20
Quiz on Wed. Amino acids (25 min)
Quiz on Friday Citric Acid Cycle (25 min)
Chapter 26: amino acid metabolism
New HW posted
Amino acid metabolism
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Amino acids function as monomers of polypeptides.
Energy metabolites.
Precursors for nitrogen-containing compounds (heme,
glutathione, nucleotides, coenzymes)
Amino acids are classified into 2 groups: essential and
nonessential
Mammals can synthesize nonessential amino acids from
metabolic precursors.
Essential amino acids must be taken in from diet.
Excess dietary amino acids are converted to common
metabolic intermediates: pyruvate, OAA, acetyl-CoA,
and -ketoglutarate.
Breakdown of amino acids
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3 stages
Deamination-the removal of the amino groupconversion to ammonia or the amino group of asp.
Incorporation of ammonia and aspartate nitrogen atoms
into urea to be exreted.
Conversion of -keto acids into common metabolic
intermediates.
Most reactions similar to those covered in other pathways.
The first step is deamination of the amino acid.
Deamination
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Most amino acids use a transamination to deminate the amino
acids.
This transfers the amino group of an a-keto acid to make a new
amino acids in reactions catalyzed by aminotransferases (aka
transaminases).
-ketoglutarate is the predominant amino group acceptor
(produces glutamate).
Amino acid + -ketoglutarate
-ketoacid + glutamate
Glutamate’s amino group is then transferred to oxaloacetate to make asp
Glutamate + OAA
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-ketoglutarate + aspartate
Glutamate dehydrogenase (GDH) main catalyst for deamination.
Glutamate + NAD(P)+ + H2O
-ketoglutarate + NH4+ + NAD(P)H
Transamination
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Aminotransferase reactions occur in 2 stages:
1.
The amino group of an amino acid is transferred to the enzyme:
Amino acid + enzyme
2.
-keto acid + enzyme-NH2
The amino group is transferred to the keto acid acceptor, ketoglutarate to form glutamate and regenerate the enzyme.
-ketoglutarate + enzyme-NH2
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enzyme + glutamate
Aminotransferases require the aldehyde-containing coenzyme,
pyridoxal-5’-phosphate (PLP) a derivative of pyridoxine (aka
vitamin B6).
PLP is attached to the enzyme via a Schiff base linkage by
condensation of the aldehyde group to thee -amino group of a
Lys within the enzyme.
PLP is converted to pyridoxamine-5’-phosphate (PMP)
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Figure 26-1ab Forms of pyridoxal-5¢-phosphate.
(a) Pyridoxine (vitamin B6) and (b) Pyridoxal-5¢-phosphate
(PLP).
Forms of pyridoxal-5¢phosphate.
(c) Pyridoxamine-5¢-phosphate (PMP) and (d) The Schiff
base that forms between PLP and an enzyme -amino
group.
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Figure 26-1cd
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Step 1: amino group acts as a nuclophile to attack the
enzyme-PLP Schiff base carbon to form an amino acid-PLPSchiff base (transamination aka trans-Schiffization).
This releases the Lys amino group and the Lys can act as a
general base catalyst.
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Enz-Lys removes the amino
acid -carbon H
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Transamination
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Can be reversed to convert an -keto acid to an amino acid
PLP functions as an electron sink.
Cleavage of any of the amino acid C atom’s 3 bonds produces a
resonance stabilized structure.
PLP can therefore be used in both transamination and
decarboxylation reactions.
Most aminotransferases accept only -ketoglutarate or
oxaloacetate as the -keto acid substrate in the second stage of
the reaction (reverse reaction).
The amino groups of most amino acids are therefore incorporated in
the formation of glutamate or aspartate.
Glu and Asp are connected by glutamate-aspartate
aminotransferase.
Glutamate + oxaloacetate
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-ketoglutarate + aspartate
Oxidative deamination of glutamate regenerates -ketoglutarate
and makes ammonia.
Ammonia and aspartate are the amino donors for urea synthesis.
Transamination
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Can be reversed to convert an -keto acid to an amino acid
PLP functions as an electron sink.
Cleavage of any of the amino acid C atom’s 3 bonds produces a
resonance stabilized structure.
PLP can therefore be used in both transamination and
decarboxylation reactions.
Most aminotransferases accept only -ketoglutarate or
oxaloacetate as the -keto acid substrate in the second stage of
the reaction (reverse reaction).
The amino groups of most amino acids are therefore incorporated in
the formation of glutamate or aspartate.
Glu and Asp are connected by glutamate-aspartate
aminotransferase.
Glutamate + oxaloacetate
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-ketoglutarate + aspartate
Oxidative deamination of glutamate regenerates -ketoglutarate
and makes ammonia.
Ammonia and aspartate are the amino donors for urea synthesis.
Glucose-Alanine Cycle
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Exception-muscle aminotransferases that accept
pyruvate as their -keto acid substrate
Produce alanine to be transported to the liver via the
bloodstream.
Once in the liver, Ala is transformed back into pyruvate
for use in gluconeogenesis.
Glucose returned to muscle cells to be degraded to
pyruvate.
During starvation, glucose formed in the liver is used by other
tissues and breaks the cycle.
Amino groups will be derived from muscle to provide glucose
for the other tissues.
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Figure 26-3 The glucose–
alanine cycle.
Oxidative demaniation
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Glutamate
dehydrogenase
(GDH) can use
either NAD+ or
NADP+ as redox
coenzyme.
Allosterically
inhibited by GTP
and NADH.
Activated by ADP,
Leu, and NAD+.
Other deamination pathways
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Gln made from glutamate and ammonia by glutamine
synthestase. N can be transported to the liver from
Gln.
Ammonia is released for urea production in the liver
mitochondria or for excretion after processing by
glutiminase.
Other deamination pathways
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Gln made from glutamate and ammonia by glutamine
synthestase. N can be transported to the liver from
Gln.
Ammonia is released for urea production in the liver
mitochondria or for excretion after processing by
glutiminase.
Other deamination pathways
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Gln made from glutamate and ammonia by glutamine
synthestase. N can be transported to the liver from
Gln.
Ammonia is released for urea production in the liver
mitochondria or for excretion after processing by
glutiminase.
Oxidative demaniation
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Glutamate
dehydrogenase
(GDH) can use
either NAD+ or
NADP+ as redox
coenzyme.
Allosterically
inhibited by GTP
and NADH.
Activated by ADP,
Leu, and NAD+.
Glu
Figure 26-5a X-Ray
structures of
glutamate
dehydrogenase
(GDH). (a) Bovine
GDH in complex with
glutamate, NADH,
and GTP.
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NADH
GTP
NADH bound at ADP effector site
Figure 26-5bX-Ray
structures of
glutamate
dehydrogenase
(GDH). (b) One
subunit of the bovine
GDH–glutamate–
NADH–GTP
complex.
Antenna
domain
Pivot helix
NADH
GTP
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Coenzyme
binding
domain
Substrate
binding
domain
NADH
Glu
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Figure 26-5c X-Ray structures of glutamate
dehydrogenase (GDH). (c) One subunit of human
apoGDH with the protein colored as and viewed
similarly to Part b.
Binding
rotates
about pivot
helix
causing
cleft to
close
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Figure 26-6 Inhibition of human glutamate
dehydrogenase (GDH) by GTP.
(50% inhibition at midpoint)
Other deamination pathways
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Nonspecific amino acid oxidases - L-amino acid
oxidase and D-amino acid oxidase.
Have FAD as redox coenzyme.
Amino acid + FAD + H2O  -keto acid + NH3 + FADH2
FADH2 + O2  FAD + H2O2
Urea Cycle
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Excess nitrogen is excreted after the metabolic breakdown of amino acids in one of
three forms:
Aquatic animals are ammonotelic (release NH3 directly).
If water is less plentiful, NH3 is converted to less toxic products, urea and uric acid.
Terrestrial vertebrates are ureotelic (excrete urea)
Birds and reptiles are uricotelic (excrete uric acid)
Urea is made by enzymes urea cycle in the liver.
The overall reaction is:
NH3+
NH3 + HCO3- + -OOC-CH2-CH-COOAsp
O
3ATP
2ADP + 2Pi + AMP + PPi
NH2-C-NH2 + -OOC-CH=CH-COOUrea
Fumarate
Urea Cycle
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2 urea nitrogen atoms come from ammonia and
aspartate.
Carbon atom comes from bicarbonate.
5 enzymatic reactions used, 2 in the mitochondria and 3
in the cytosol.
NH3+
NH3 + HCO3- + -OOC-CH2-CH-COOAsp
O
3ATP
2ADP + 2Pi + AMP + PPi
NH2-C-NH2 + -OOC-CH=CH-COOUrea
Fumarate
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Carbamoyl phosphate synthetase
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Carbamoyl phosphate synthetase (CPS) catalyzes the
condensation and activation NH3 and HCO3- to form carbomyl
phosphate (first nitrogen containing substrate).
Uses 2 ATPs.
O
2ATP + NH3 + HCO3-  NH2-C-OPO3- + 2ADP + 2Pi
Carbamoyl phosphate
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Eukaryotes have 2 types of CPS enzymes
Mitochondrial CPSI uses NH3 as its nitrogen donor and participates in urea
biosynthesis.
Cytosolic CPSII uses glutamine as its nitrogen donor and is involved in
pyrimidine biosynthesis.
Figure 26-8 The
mechanism of action of
CPS I.
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1.
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CPSI reaction has 3 steps
Activation of HCO3- by ATP to form
carboxyphosphate and ADP.
2.
Nucelophilic attack of NH3 on
carboxyphosphate, displacing the
phsophate to form carbamate and
Pi.
3.
Phosphorylation of carbamate by the
second ATP to form carbamoyl
phosphate and ADP
The reaction is irreversible.
Allosterically activated by Nacetylglutamate.
Figure 26-9 X-Ray structure of
E. coli carbamoyl phosphate
synthetase (CPS).
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E. coli has only one CPS
(homology to CPS I and CPS II)
Heterodimer (inactive).
Allosterically activated by ornithine
(heterotetramer of (4).
Small subunit hydrolyzes Gln and
delivers NH3 to large subunit.
Channels intermediate of two
reactions from one active site to the
other.
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Ornithine transcarbomylase
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Transfers the carbomoyl group of carbomyl phosphate to ornithine
to make citrulline
Reaction occurs in mitochondrion.
Ornithine produced in the cytosol enters via a specific transport
system.
Citrulline is exported from the mitochondria.
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Arginocuccinate Synthetase
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2nd N in urea is incorporated in the 3rd reaction of the urea cycle.
Condensation reaction with citrulline’s ureido group with an Asp
amino group catalyzed by arginosuccinate synthetase.
Ureido oxygen is activated as a leaving group through the
formation of a citrulyl-AMP intermediate.
This is displaced by the Asp amino group to form
arginosuccinate.
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Figure 26-10 The mechanism of action of
argininosuccinate synthetase.
Arigininosuccinase and Arginase
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Argininosuccinse catalyzes the elimination of Arg from
the the Asp carbon skeleton to form fumurate.
Arginine is the immediate precursor to urea.
Fumurate is converted by fumarase and malate
dehydrogenase to to form OAA for gluconeogenesis.
Arginase catalyzes the fifth and final reaction of the
urea cycle.
Arginine is hydrolyzed to form urea and regenerate
ornithine.
Ornithine is returned to the mitochondria.
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1. Carbamoyl
phosphate
synthetase (CPS)
2. Ornithine
transcarbamoylase
3. Argininosuccinate
synthetase
4. Arginosuccinase
5. Arginase
Regulation of the urea cycle
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Carbamoyl phosphate synthetase I is allosterically
activated by N-acetylglutamate.
N-acetylglutamate is synthesized from glutamate and acetylCoA by N-acetylglutamate synthase, it is hydrolyzed by a
specific hydrolase.
Rate of urea production is dependent on [N-acetylglutamate].
When aa breakdown rates increase, excess nitrogen must be
excreted. This results in increase in Glu through
transamination reactions.
Excess Glu causes an increase in N-acetylglutamate which
stimulates CPS I causing increases in urea cycle.
Metabolic breakdown of amino
acids
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Degradation of amino acids converts the to TCA cycle
intermediates or precursors to be metabolized to CO2, H2O,
or for use in gluconeogenesis.
Aminoacids are glucogenic, ketogenic or both.
Glucogenic amino acids-carbon skeletons are broken down
to pyruvate, -ketoglutarate, succinyl-CoA, fumarate, or
oxaloacetate (glucose precursors).
Ketogenic amino acids, are broken down to acetyl-CoA or
acetoacetate and therefore can be converted to fatty acids or
ketone bodies.