Nitrogen Metabolism
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•
•
•
•
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Protein degradation and turnover
Amino acid degradation and urea cycle
Nitrogen cycle
Nitrogen fixation
Amino acid biosynthesis
Amino acid derivatives
How Much Protein?
• A 70 kg person (154 lb) typically consumes 100 g
protein per day
• To stay in nitrogen balance that person must
excrete 100 g of N products per day
• The body makes 400 g of protein per day and 400
g are broken down
• 300 g of amino acids recycled into new protein,
100 g are degraded
• Total protein = 500 g/day, 400 g degraded, 400
resynthesized and 100 g catabolized
Characteristic of Proteins in Cells
• Synthesized and degraded constantly Turnover
• Turnover may be minutes, weeks or longer
• Synthesis requires essential and non essential
amino acids
• Degradation is programmed and regulated
• Control point enzymes most labile; constitutive
most stable
• Nutritional state and hormones affect
degradation rates (glucocorticoids, insulin, etc.)
The half-life of proteins is determined by
rates of synthesis and degradation
dC
Rate of Turnover =
dt
= KS - KDC
A given protein is synthesized at a constant rate KS
A constant fraction of active molecules are destroyed per
unit time
KS is the rate constant for protein synthesis; will
vary depending on the particular protein
C is the amount of Protein at any time
KD is the first order rate constant of enzyme degradation,
i.e., the fraction destroyed per unit time, also
depends on the particular protein
Steady-state is achieved when the amount of protein
synthesized per unit time equals the amount being destroyed
dC
= 0 KDC = KS
dt
t 1/2 =
0.693
KD
C
Protein
concentration
(enzyme activity)
Stop protein synthesis,
measure rate of decay
Hours after stopping synthesis
Steps in Protein Degradation
Transformation to a degradable form
(Metal oxidized, Ubiquination, N-terminal residues, PEST sequences)
Lysosomal Digestion
ATP
26S Proteasome digestion
AMP + PPi
Proteolysis to peptides
KFERQ
7  type, 7  type
subunits
8 residue fragments
Ubiquination
N-end rule:
PEST:
DRLKF: 2-3 min
AGMSV: > 20 hr
Rapid degradation
Glycine at C terminal of Ubiquitin
Ubiquitin
COOATP
Ubiquitin activating enzyme
HS
AMP + PPi
E1
O
C S
Activation
of Ubiquitin
Ubiquitin conjugating enzyme
20 or more per cell
NH3+
E1
HS
E2
3
HS E1
C
S
Ubiquitin
ligase
E2
O
C N
O
C
Poly Ubiquitin
Ubiquination
Page 1075
SH
NH3+
H3N+
O
3
E2
ATP
NH
AMP + PPi
Degraded
protein
E3
O
N C
O
N C
Ubiquitinspecific proteases
(26S proteasome)
+ Ubiquitin
Cervical Cancer
Human Papilloma virus (HPV)
Activates the E3 that catalyzes ubiquination of
p53 tumor suppressor and DNA repair enzymes
(occurs in 90% of cervical cancers)
Mutated DNA is unchecked and allowed to replicate
P472
19S
20S
Catalysis
in beta
19S
Subunits
26S Proteasome
(2000 kD)
7 alpha
7 beta
Opening for ubiquinated
protein to enter
8-residue peptides diffuse out
Amino Acids
Amine Group
Glutamate
Urea
Carbon Skeleton
Biosynthesis
Amino Acids
Amino Acid
Derivatives
Degradation
CO2 + H2O
-Ketoglutarate-Glutamate
COOC=O
CH2
CH2
COO-Kg
Amine group acceptor
COO+
H3N-C-H
CH2
Amine group donor CH2
COOL-glutamate
AA1 + -KG
-ketoacid + glutamate
acceptor
donor
Amino transferases
Requires pyridoxal-5’-phosphate
CH2OH
Vitamin B6
HO
H 3C
CH2OH
Pyridoxine
N
O
Cofactor (N acceptor)
C
HO
H3 C
H
CH2OP
N
CH2NH2
Cofactor (N donor)
HO
H3 C
Pyridoxal-5’-PO4
CH2OP
N
Pyridoxamine-PO4
Alanine-Pyruvate Aminotransferase
COO+
H3N-C-H +
CH3
O
C
HO
H3 C
COOC=O
CH2
CH2
COOforward
reverse
H
HO
H3C
CH2OP
N
O
C
CH2NH2
CH2OP
N
COO+
H3N-C-H
C=O +
CH2
CH3
CH2
COO-
COO-
HO
H3 C
H
CH2OP
N
Mechanism
Alanine
In
Glutamate
Pyruvate -Ketoglutarate
Out
Enz-CHO
(E-B6-al)
Enz-NH2
(E-B6-am)
In
Enz-NH2
Out
Enz-CHO
Ordered Ping-Pong Mechanism
Glutamate Metabolism
COO+
H3N-C-H
CH2 + NAD(P)+ + H2O
CH2
Glutamate
COOdehydrogenase
COOC=O
CH2 + NAD(P)H + H+
+ NH4+
CH2
COO-
Urea cycle
Forward Reaction
Reverse Reaction
specific for glutamate
specific for -ketoglutarate
requires NAD+
requires NADPH
delivers NH4+ to urea cycle
Fixes NH4+, prevents toxicity
Glutamine Metabolism
COOCOO+
+
H3N-C-H
H3N-C-H
+ ATP + NH4+
CH2 + ADP + Pi
CH2
Glutamine
CH2
CH2
L-glutamine
Synthetase
C=O
COONH2
H2O
COOGlutaminase
+
H3N-C-H
COO
CH2
+
H3N-C-H
CH2
+ NH4+
Glutamate-PO4
CH2
C=O
intermediate
CH2
=
OPO3
Urea
COO-
Overall Scheme Using Alanine as an Example
Amino transferase with pyridoxal-5’-PO4
Alanine
Pyruvate
-ketoglutarate
glutamate
Glutamate dehydrogenase
with NAD+
NH4+
Glutaminase with H2O
glutamine
Urea
Glutamate and glutamine are the only donors
of NH3 to the Urea Cycle
The Urea Cycle
1. Occurs in the liver mitochondria and cytosol
2. Starts with carbamoyl-PO4
3. Ends with arginine
4. Requires aspartate
5. Requires 3 ATPs to make one urea
Synthesis of Carbamoyl-PO4
NH4+ + HCO3- + 2 ATP
O
H2N
O
C ~ O-P-O
+ 2 ADP + Pi
O
High energy bond
Carbamoyl phosphate Synthetase I
Citrulline
Carbamoyl-PO4
Urea
Cycle
Ornithine
+
NH3
CH2
CH2
CH2
HC
COOH3N
Arginine
H2O
O
C
H2 N
NH2
Urea
Aspartate
ATP
Argininosuccinate
NH2
+
H2N=C
NH
CH2
CH2
CH2
HC
COOH3N
Reactions of Urea Cycle
COOH3N+-C-H
O
CH2
CH2
CH2
+ NH
3
COOH3N+-C-H
+
H2N
C
CH2
CH2
CH2
NH
OPO3
Carbamoyl-PO4
O=C
Ornithine
CH2
CH2
CH2
NH
O=C
NH2
H3N+-C-H
COO+
Citruline
COO-
COOH3
+ OPO3=
NH2
Mitochondria
N+-C-H
Cytosol
ATP
CH2
+
H-C-NH3
COO-
L-Aspartate
ADP + Pi
CH2
CH2
COO- CH
2
CH2
NH
H-C-N =C
COO- NH2
Argininosuccinate
Cytosol
COOH3N+-C-H
COOCH2
CH2
CH2
COO-
CH2
CH2
NH
CH2
CH2
NH
CH
H2N+ =C
H-C-N =C
COO-
COOH3N+-C-H
NH2
+
HC
COOFumarate
NH2
L-Arginine
COO-
COO-
COO-
CH2
CH2
CH2
H-C-NH3
C=O
H C-OH
COO-
COO-
+
L-Aspartate
Oxaloacetate
COOL-Malate
COOH3N+-C-H
COOH3N+-C-H
CH2
H2O
CH2
CH2
CH2
NH
CH2
CH2
+ NH3
NH2
Ornithine
H2N+ =C
O
+
C
H2N
NH2
Urea
L-Arginine
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