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Nitrogen Metabolism (and Related Topics)
• Amino Acid Metabolism (Nitrogen metabolism)
• Folate Metabolism (“One-Carbon pathways”)
• Nucleotide Metabolism
Dr. Robert Lyons
Assistant Professor, Biological Chemistry
Director, DNA Sequencing Core
There are also PDF’s of class
handouts with supplemental
information available in the
table of contents for this course.
Supplementary study material on the Web:
http://seqcore.brcf.med.umich.edu/mcb500
Amino Acid met abolism
A mino acids
Folat e met abolism
Met hylene
THF
Glu, Gln,
Asp , NH3
Met
Cycle
Urea
oxaloac et at e
Pu r in e s
Uric Acid
DNA
RNA
Py r im id in e s
( energy )
f um arat e
TCA Cycle
Nucleic Acid met abolism
Protein Degradation:
• Endogenous proteins degrade continuously
- Damaged
- Mis-folded
- Un-needed
• Dietary protein intake - mostly degraded
Nitrogen Balance - expresses the patient’s current
status - are they gaining or losing net Nitrogen?
Transaminases
Collect Amines
General react ion overview:
H
H
R1 C COO
NH2
amino
acid
(-) +
(-)
R2 C COO
O
-keto
acid
(typically
alpha-ketoglutarate)
R1 C
O
COO
-keto
acid
(-) + R C COO(-)
2
NH2
amino
acid
(typically glutamate)
Det ails of react ion mechanism:
-keto
amino
acid
H
R C
(-)
COO
NH2
+
O
CH
H2O
P OCH2
OH
+ CH3
N
H
pyridoxal
phosphate
acid
H+
H
R C
(-)
COO
N
CH
P OCH2
OH
+ CH3
N
H
R C
N
COO(-)
R C
N
HCH
CH
P OCH2
OH
N CH3
H
COO(-)
P OCH2
OH
+
N CH3
H
(-)
R C COO
O
+
NH2
HCH
P OCH2
OH
+
N CH3
H
pyridoxamine
phosphate
Transfer the amine back to an acceptor -keto acid
P O CH
NH
O
H CH
CH
OH
CH
N
H
pyridoxamine
phosphate
+
R
C
P O CH
COO
O
-keto
acid
H
OH
CH
N
H
pyridoxal
phosphate
+
R
C
NH
amino
acid
COO
In peripheral tissues, transaminases tend to form
Glutamate when they catabolize amino acids
In other words, alpha-ketoglutarate is the preferred
acceptor, and Glutamate is the resulting amino acid:
Some amino acid + -ketoglutarate  some alpha keto acid + Glutamate
Glutamate can donate its amines to
form other amino acids as needed
A specific example - production of Aspartate in liver
(described a few slides from now):
Glutamate + oxaloacetate  -ketoglutarate + aspartate
Ge t t ing A m ine s Int o t he Liv e r
Glutamate
Dehydrogenase:
Glutamine
Synthetase:
H
OCCHCH C CO
NH
glutamate
NAD(P)
H
OOC C CHCH COO
NH
glut amat e
(mito)
NAD(P)H
ATP NH
OCCHCH C CO
O
-ketoglutarate
ADP P
+
NH
ammonia
H
O
OOC C CH CHC
NH
NH
glut amine
.
In t he Liv e r: Pre curse rs f or Ure a Cy cle
Glut amine is hydrolyzed t o glut amat e and ammonia:
H
O
OOC C CH CHC
NH
NH
glut amine
H
O
OOC C CH CHC
OH
NH
HO
NH3
glut amat e
Ammonia can also be formed by the glutamate dehydrogenase reaction
and several other reactions as well.
Glut amat e donat es it s amino group t o f orm aspart at e:
Glut amat e-aspartat e aminot ransferase:
H
OCCHCH C CO
NH
Glutamate
.
+
H
OCCHC CO
O
oxaloacetate
OCCHCHC CO
O
-keto
glutarate
+
H
OCCH C CO
NH
aspartate
2ATP + HCO3+ NH3
1
Carbamoy l
phosphat e
O
(-)
NH2C OPO3
Pi
2
2ADP + Pi
H
(-)
OOC C
H
(-)OOC C
CH2CH2CH2NH3(+)
NH
(+)3
O
CH2CH2CH2NH C NH2
NH
(+)3
Ornit hine
Cit rulline
Liver mitochondrion
Liver cyt oplasm
H
(-)
OOC C
H
(-)
OOC
+)
CH2CH2CH2NH(
3
C
NH
(+)3
O
CH2CH2CH2NH C NH2
NH
(+)3
Ornit hine
O
NH2 C NH2
Urea
Cit rulline
ATP
3
5
H
(-)O CCH C CO(-)
2 2
2
NH2
aspartate
AMP + PPi
H2O
H
(-)OOC C
CH2CH2CH2NH C NH2
NH
NH
(+) 2
(+)3
H
(-)
OOC C
NH
(+)3
A rginine
4
(-)
(-)O C C H
2 H C CO2
Fumarat e
H
(-)O CCH C CO(-)
2 2
2
NH2
+)
CH2CH2CH2NH C NH(
2
A rgininosuccinat e
Carb am o y l p h o s p h at e sy n t h e t as e I
O
HO C O
NH
ATP
bicarbonate
ADP
ATP
O
HO C O P
O
HO C NH
carbonyl
phosphate
carbamate
Pi
O
P O C NH
ADP
carbamoyl
phosphate
O r n it h in e T r a n s c a r b a m o y la s e
Carb am o y l p ho s p hat e
O
NH C OPO
P
H
OOC C
H
CH CH CHNH
CH NH
NH
O r n it h in e
OOC C
NH
C it r u llin e
O
CH CH CH NH C NH
A r g in in o s u c c in a t e s y n t h e t a s e
H
(-)O CC C CO(-)
2
2 2
H
NH2
aspartate
H
(-)
OOC C
NH
(+)3
H
O
CH2CH2CH2NH C NH2
(-)
OOC C
NH
(+)3
Cit rulline
ATP
AMP + PPi
H
(-)O CCH C CO(-)
2
2 2
NH2
(+)
CH2CH2CH2NH C NH2
A rgininosuccinat e
A r g in in o s u c c in a t e ly a s e
H
(-)
OOC C
H
(-)O CCH C CO(-)
2
2 2
NH2
(+)
CH2CH2CH2NH C NH2
H
(-)
OOC C
CH2CH2CH2NH C NH2
NH2
NH3
(
+)
(+)
NH
(+)3
Argininosuccinat e
(-)O C C CH CO(-)
2
2 H
Fumarat e
Arginine
A r g in a s e
H
H
(-)
OOC C
(-)
OOC C
CH2CH2CH2NH C NH2
NH2
NH3
(
+)
(+)
A rginine
NH
(+)3
H2 O
O
NH2C NH2
Urea
CH2CH2CH2NH(3+)
Ornit hine
2ATP + HCO3+ NH3
1
Carbamoy l
phosphat e
O
(-)
NH2C OPO3
Pi
2
2ADP + Pi
H
(-)
OOC C
H
(-)OOC C
CH2CH2CH2NH3(+)
NH
(+)3
O
CH2CH2CH2NH C NH2
NH
(+)3
Ornit hine
Cit rulline
Liver mitochondrion
Liver cyt oplasm
H
(-)
OOC C
H
(-)
OOC
+)
CH2CH2CH2NH(
3
C
NH
(+)3
O
CH2CH2CH2NH C NH2
NH
(+)3
Ornit hine
O
NH2 C NH2
Urea
Cit rulline
ATP
3
5
H
(-)O CCH C CO(-)
2 2
2
NH2
aspartate
AMP + PPi
H2O
H
(-)OOC C
CH2CH2CH2NH C NH2
NH
NH
(+) 2
(+)3
H
(-)
OOC C
NH
(+)3
A rginine
4
(-)
(-)O C C H
2 H C CO2
Fumarat e
H
(-)O CCH C CO(-)
2 2
2
NH2
+)
CH2CH2CH2NH C NH(
2
A rgininosuccinat e
Urea Cycle Connect s t o TCA Cycle
Ornithine
H
(-)O CCH C CO(-)
2
2 2
NH2
Citrulline
Aspartate
Urea
Urea Cycle
Arginine
Argininosuccinate
Oxaloacetate
Malate
H
-)
(-)OC C C
CO(
2
2 H
Fumarate
TCA Cycle
-Ketoglutarate
Citrate
Ge t t ing A m ine s Int o t he Liv e r
Glutamate
Dehydrogenase:
Glutamine
Synthetase:
H
OCCHCH C CO
NH
glutamate
NAD(P)
H
OOC C CHCH COO
NH
glut amat e
(mito)
NAD(P)H
ATP NH
OCCHCH C CO
O
-ketoglutarate
ADP P
+
NH
ammonia
H
O
OOC C CH CHC
NH
NH
glut amine
2ATP + HCO3+ NH3
1
Carbamoy l
phosphat e
O
(-)
NH2C OPO3
Pi
2
2ADP + Pi
H
(-)
OOC C
H
(-)OOC C
CH2CH2CH2NH3(+)
NH
(+)3
O
CH2CH2CH2NH C NH2
NH
(+)3
Ornit hine
Cit rulline
Liver mitochondrion
Liver cyt oplasm
H
(-)
OOC C
H
(-)
OOC
+)
CH2CH2CH2NH(
3
C
NH
(+)3
O
CH2CH2CH2NH C NH2
NH
(+)3
Ornit hine
O
NH2 C NH2
Urea
Cit rulline
ATP
3
5
H
(-)O CCH C CO(-)
2 2
2
NH2
aspartate
AMP + PPi
H2O
H
(-)OOC C
CH2CH2CH2NH C NH2
NH
NH
(+) 2
(+)3
H
(-)
OOC C
NH
(+)3
A rginine
4
(-)
(-)O C C H
2 H C CO2
Fumarat e
H
(-)O CCH C CO(-)
2 2
2
NH2
+)
CH2CH2CH2NH C NH(
2
A rgininosuccinat e
C P S I is S t im u la t e d b y N A G
H
H
OOC
C
N -a c e t y l
CH CH C
NH
O
OH
g lu t a m a t e
+
Co A - C
O
OOC
C
CH CH C
OH
NH
g lu t a m a t e
s y n t h e t as e
O
C O
CH
CH
N - a c e t y l g lu t a m a t e
a c e t y l Co A
( N A G)
( r e p e a t in g t h e f ig u r e f r o m p a g e 3 o f y o u r h an d o u t )
O
HO C O
ATP
bicarbonate
O
HO C O P
ADP
carbonyl
phosphate
NH
O
HO C NH
Pi
ATP
carbamate
O
P O C NH
ADP
carbamoyl
phosphate
-Muscle
(Amines)
Glucose
Pyruvate
Glutamate
Amino acids
-ketoglutarate
Alanine
blood
transport
Alanine -ketoglutarate
Glucose
Pyruvate
Glutamate
Live r
Urea
NH
Complicating the picture: Other tissues may be involved
Muscle:
Amino acids:
Transamination
Deamination
Alanine
purine
deamination:
(+)
Glutamate
NH4
Glutamine
Intestine:
Glutamine
(+)
Alanine NH4
Glutamine
Citrulline
Kidney:
Citrulline
NH3
(+)
NH4
Arginine
Liver:
Arginine
Glutamine
(+)
NH4
Alanine
Glu
Aspartate
Urea
Why is Ammonia Toxic?
Why is Ammonia Toxic?
• Possible neurotoxic effects on
glutamate levels (and also GABA)
(due to shifting equilibria of reactions
involving these compounds)
Why is Ammonia Toxic?
• Possible neurotoxic effects on
glutamate levels (and also GABA)
(due to shifting equilibria of reactions
involving these compounds)
• Possible metabolic/energetics effects:
- alpha-ketoglutarate levels
- glutamate levels
- glutamine
2ATP + HCO3+ NH3
1
Carbamoy l
phosphat e
O
(-)
NH2C OPO3
Pi
2
2ADP + Pi
H
(-)
OOC C
H
(-)OOC C
CH2CH2CH2NH3(+)
NH
(+)3
O
CH2CH2CH2NH C NH2
NH
(+)3
Ornit hine
Cit rulline
Liver mitochondrion
Liver cyt oplasm
H
(-)
OOC C
H
(-)
OOC
+)
CH2CH2CH2NH(
3
C
NH
(+)3
O
CH2CH2CH2NH C NH2
NH
(+)3
Ornit hine
O
NH2 C NH2
Urea
Cit rulline
ATP
3
5
H
(-)O CCH C CO(-)
2 2
2
NH2
aspartate
AMP + PPi
H2O
H
(-)OOC C
CH2CH2CH2NH C NH2
NH
NH
(+) 2
(+)3
H
(-)
OOC C
NH
(+)3
A rginine
4
(-)
(-)O C C H
2 H C CO2
Fumarat e
H
(-)O CCH C CO(-)
2 2
2
NH2
+)
CH2CH2CH2NH C NH(
2
A rgininosuccinat e
Inherited Defects of Urea Cycle Enzymes: Diagnosis
Defects are diagnosed based on the metabolites seen
in the blood and/or urine.
CPSD
OTCD
ASD
ALD
AD
No elevation except ammonia; diagnosed by elimination.
Elevated CP causes synthesis of Orotate
Elevated citrulline
Elevated argininosuccinate
Elevated arginine
2ATP + HCO3+ NH3
1
Carbamoy l
phosphat e
O
(-)
NH2C OPO3
Pi
2
2ADP + Pi
H
(-)
OOC C
H
(-)OOC C
CH2CH2CH2NH3(+)
NH
(+)3
O
CH2CH2CH2NH C NH2
NH
(+)3
Ornit hine
Cit rulline
Liver mitochondrion
Liver cyt oplasm
H
(-)
OOC C
H
(-)
OOC
+)
CH2CH2CH2NH(
3
C
NH
(+)3
O
CH2CH2CH2NH C NH2
NH
(+)3
Ornit hine
O
NH2 C NH2
Urea
Cit rulline
ATP
3
5
H
(-)O CCH C CO(-)
2 2
2
NH2
aspartate
AMP + PPi
H2O
H
(-)OOC C
CH2CH2CH2NH C NH2
NH
NH
(+) 2
(+)3
H
(-)
OOC C
NH
(+)3
A rginine
4
(-)
(-)O C C H
2 H C CO2
Fumarat e
H
(-)O CCH C CO(-)
2 2
2
NH2
+)
CH2CH2CH2NH C NH(
2
A rgininosuccinat e
C P S I is S t im u la t e d b y N A G
H
H
OOC
C
N -a c e t y l
CH CH C
NH
O
OH
g lu t a m a t e
+
Co A - C
O
OOC
C
CH CH C
OH
NH
g lu t a m a t e
s y n t h e t as e
O
C O
CH
CH
N - a c e t y l g lu t a m a t e
a c e t y l Co A
( N A G)
( r e p e a t in g t h e f ig u r e f r o m p a g e 3 o f y o u r h an d o u t )
O
HO C O
ATP
bicarbonate
O
HO C O P
ADP
carbonyl
phosphate
NH
O
HO C NH
Pi
ATP
carbamate
O
P O C NH
ADP
carbamoyl
phosphate
2ATP + HCO3+ NH3
1
Carbamoy l
phosphat e
O
(-)
NH2C OPO3
Pi
2
2ADP + Pi
H
(-)
OOC C
H
(-)OOC C
CH2CH2CH2NH3(+)
NH
(+)3
O
CH2CH2CH2NH C NH2
NH
(+)3
Ornit hine
Cit rulline
Liver mitochondrion
Liver cyt oplasm
H
(-)
OOC C
H
(-)
OOC
+)
CH2CH2CH2NH(
3
C
NH
(+)3
O
CH2CH2CH2NH C NH2
NH
(+)3
Ornit hine
O
NH2 C NH2
Urea
Cit rulline
ATP
3
5
H
(-)O CCH C CO(-)
2 2
2
NH2
aspartate
AMP + PPi
H2O
H
(-)OOC C
CH2CH2CH2NH C NH2
NH
NH
(+) 2
(+)3
H
(-)
OOC C
NH
(+)3
A rginine
4
(-)
(-)O C C H
2 H C CO2
Fumarat e
H
(-)O CCH C CO(-)
2 2
2
NH2
+)
CH2CH2CH2NH C NH(
2
A rgininosuccinat e
Clinical Management of Urea Cycle Defects
• Dialysis to remove ammonia
• Provide the patient with alternative ways to excrete
nitrogenous compounds:
* Intravenous sodium benzoate or phenylacetate
* Supplemental arginine
carbamoyl phosphate
Excreted by kidney
O
NH2C NH2
Ornithine
Citrulline
ATP
Urea
AMP+PP i
HO
2
Arginine
Aspartate
XASD
Argininosuccinate
Excreted by kidney
Dietary
Arginine
• Levulose - acidifies the gut
• Low protein diet
XALD
Degrading the Amino Acid Carbon Backbone
Easily-degraded products after transamination:
H
(-)O CCH C CO(-)
(-)O CCH C CO(-)
2
2 2
2
2 2
t ransaminat ion
NH3
O
(+)
oxaloacetate
aspartate
H
(-)O CCHCH C CO(-)
(-)O CCH CH C CO(-)
2
2
2 2 2
2 2 2
t
ransaminat
ion
O
NH
(+)3

-ketoglutarate
glutamate
H
-)
CH3 C CO(
2
t ransaminat ion
NH
(+)3
alanine
-)
CH3 C CO(
2
O
pyruvate
We also already know how to degrade Glutamine:
glutaminase
Glutamine ----------------> glutamate + ammonia
…and by analogy, how to degrade Asparagine:
asparaginase
Asparagine ---------------> aspartate + ammonia
Amino Acids are categorized as ‘Glucogenic’
or ‘ketogenic’ or both.
Many amino acids are purely glucogenic:
Glutamate, aspartate, alanine, glutamine,
asparagine,…
Some amino acids are both gluco- and ketogenic:
Threonine, isoleucine, phenylalanine,
tyrosine, tryptophan
The only PURELY ketogenic Amino Acids:
leucine, lysine
H
(-)
OOC C
CH2CH2CH2NH C NH2
NH
NH
(+)2
(+)3
Arginine
Urea ( via
t he urea cy cle)
H
(-)
OOC C
Amino acids with 5-carbon
backbones tend to form
-ketoglutarate
NH
(+)3
CH2CH2CH2NH(3+)
Ornit hine
-ket oglut arat e
glut amat e
H
(-)
OOC C
CH2CH2C
NH
(+)3
(-)
OOC
O
(+)
N
H
Proline
H
glut amat e - 5 - semialdehyde
(+)
NAD(P)
NA D(P)H
H
(-)
OOC C
CH2CH2C
NH
(+)3
glut amat e
 - ket oglut arat e
H
(-)
OOC C
O
OH
O
CH2CH2C
NH
(+)3
NH3
H2O
glut amine
NH2
Degradation and Biosynthesis of Serine and Glycine
(+)
NAD
Glycine
Synthase:
H
(-)
OOC C
NH
(+)3
CO2
+
(+)
NH4
H
THF
Glycine
Serine
Hydroxymethyltransferase:
NADH
N5-N10- methylene THF
(-)
OOC C 2 NH3
(+)
H
(-)
OOC CH
NH
(+)3
CH2OH
THF
Serine
N5-N10- methylene THF
Gly cine
(+)
NH4
H2O
Serine
Dehydratase:
(-)
OOC CH (
NH
+)3
CH2OH
Serine
(-)
OOC
C
CH2
(+)
NH3
(-)
OOC
C
CH3
(+)
NH2
H2O
(-)
OOC
C O
CH3
H
CH S CH CH C COO
CH
NH
H
CH S CH CH C COO
NH
ATP +
HO
Met hionine
PPi +
Pi
O Ad en in e
H HH
H OH OH
S-A denosy l Met hionine
Met h yl a cce pt o r
te t rah yd ro fo lat e
Bi os ynt h et ic
Me t hy lat io n
re act io n
N5 me th yl
t et rah yd ro fo la te
Methionine Cycle
And Biological
Methyl Groups
*
se e e xam pl es
Me t hyl at ed acc ep to r
H
HS CH CH C COO
CH
NH
H
HS CH CH C COO
NH
O Ad en in e
H HH
H OH OH
Homoc y st eine
H
HO CH C COO
NH
Serine
H
HS CH C COO
NH
Cy st eine
(remainder of
homocysteine
degraded
for energy)
S-Adenosy l Homoc yst eine
Phenylalanine and Tyrosine
(Normal pat h shown in black, pat hological react ion shown in red)
Tetrahydrobiopterin + O2
Dihydrobiopterin + H2O
(+)
NH 3
CH2
CH
(-)
CH2
Enzyme:
Phenylalanine
hydroxylase
Phenylket onuria
(no phenylalanine
hydroxylase)
(-)
COO
Phenylpyruvat e
CH
(-)
COO
Tyrosine
Homogent isat e
O
C
NH 3
HO
COO
Phenylalanine
CH2
(+)
Deficiency:
Alkaptonuria
“Ochronosis”
Enz yme:
homogent isat e
dioxy genase
(you don’ t need t o
know t he rest )
Branched Chain Amino Acids
Isoleucine
CH3CH2CH
CH
Leucine
(-)
COO
CH3 NH3
(+)
CH3CH CH 2
CH
CH3
NH
(+)3
Valine
(-)
COO
-KG
-KG
CH3CH
CH
CH3 NH3
(+
)
(-)
COO
-KG
--- ------------ - Transaminat ion -------- -------Glu
CH3CH2CH
C
CH3
Glu
(-)
COO
O
CH3CHCH 2
C
CH3
O
+
(-)
COO
Glu
CH3CH
C
CH3 O
NAD,+CoASH
NAD,+CoASH
NAD, CoASH
(-)
COO
--- Branched-chain -ket o acid dehydrogenase - -NADH +
CO
CH3CH2CH
CH3
C
O
S-CoA
NADH + CO2
NADH + CO2
2
CH3CHCH 2
C
CH3
O
S-CoA
CH3CH
C
S-CoA
CH3 O
( cont inues on t o degradat ion pat h similar t o -oxidat ion of fatt y acids)
Synt hesis of Bioact ive Amines
(+)
NH 3
HO
CH 2
CH
Tyrosin e
(-)
COO
HO
Tyrosine
hydroxy lase
HO
HO
(+)
CH 2CH 2NH 3
HO
HO
CH 2
(-)
COO
CH
Dih ydroxyp heny lalanin e
( L-DOPA)
HO
OH
(+)
CH CH 2NH 3
H
Dop am ine
(+)
NH 3
HO
No repine phrine
HO
OH
CH CH 2 N CH 3
H
H
Ep ineph rine
Synthesis of Bioactive Amines
CH 2
N
CH
NH
(+)3
Tryp t o ph an
(-)
COO
HO
Tryptophan
hy droxylase
CH 2
CH
N
NH
(+)3
5 -hyd roxyt ryp t o ph an
PLP-de pe ndent
de cvarboxylat ion
(-)
COO
CO2
HO
CH 2
NAD+
N
Sero t o nin
CH 2
NH
(+)3
Synthesis of Bioactive Amines
(-)
COO CH 2 CH 2 CH
(-)
COO
NH
(+)3
Glut amat e
N
N
H
CH 2
CH
NH
(+)3
Hist idine
Glutam ate
de carboxylase
( PLP-depe ndent )
(-)
COO CH 2CH 2 CH2 NH 3
(+)
(COO)
Hist idine
de carboxylase
( PLP-de pe ndent )
-amin ob ut yric acid
(GABA)
N
N
H
CH 2
CH2
Hist amine
NH
(+)3
NON-Essential Amino Acids:
Glutamate, aspartate, alanine, glutamine, asparagine,
(proline), glycine, serine (cysteine, tyrosine)
Essential Amino Acids:
Arginine (!), phenylalanine, methionine, histidine,
Isoleucine, leucine, valine, threonine, tryptophan, lysine