nitrogen metabolism
DESCRIPTION
Nitrogen Metabolism. 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 - PowerPoint PPT PresentationTRANSCRIPT
Nitrogen Metabolism
• 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
A given protein is synthesized at a constant rate KS
A constant fraction of active molecules are destroyed per unit time
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
KS is the rate constant for protein synthesis; will vary depending on the particular protein
Rate of Turnover = dCdt = KS - KDC
Steady-state is achieved when the amount of protein synthesized per unit time equals the amount being destroyed
dCdt
= 0 KDC = KS t 1/2 = 0.693
KD
Proteinconcentration(enzyme activity)
Hours after stopping synthesis
C
Stop protein synthesis,measure rate of decay
Steps in Protein Degradation
Transformation to a degradable form(Metal oxidized, Ubiquination, N-terminal residues, PEST sequences)
Lysosomal Digestion 26S Proteasome digestion
Proteolysis to peptides
Ubiquination
ATP
AMP + PPi
KFERQ8 residue fragments
7 type, 7 type subunits
N-end rule: DRLKF: 2-3 min AGMSV: > 20 hrPEST: Rapid degradation
COO-Ubiquitin
Glycine at C terminal of Ubiquitin
SCO
E1
HSATP
AMP + PPi
E1
HS
HS E1
E2H3N+
NH3+
NH3+
N
NCO
N CO
CO
ATP
AMP + PPi
Ubiquitin-specific proteases(26S proteasome)
Degradedprotein + Ubiquitin
Ubiquination
Ubiquitin activating enzyme
Activationof Ubiquitin
Ubiquitin conjugating enzyme 20 or more per cell
SCO
E23
E2 SH3
Ubiquitin ligase
E3
Page 1075
CPoly Ubiquitin
NH
O
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
26S Proteasome (2000 kD)
Opening for ubiquinated protein to enter
20S
19S
19S
7 alpha7 betaSubunits
Catalysis in beta
8-residue peptides diffuse out
Amino Acids
Amine Group
Glutamate
Urea
Carbon Skeleton
DegradationBiosynthesis
CO2 + H2OAmino Acids
Amino Acid Derivatives
COO-
C=OCH2
COO-
CH2
H3N-C-HCOO-
CH2
COO-
CH2
+
-Ketoglutarate-Glutamate
Amine group acceptor
Amine group donor
AA1 + -KG -ketoacid + glutamate
Amino transferasesRequires pyridoxal-5’-phosphate
-Kg L-glutamate
acceptor donor
N
CH2OPHO
H3C
CH
O
N
CH2OHHO
H3C
CH2OHVitamin B6
Pyridoxine
Cofactor (N acceptor)
Pyridoxal-5’-PO4
N
CH2OPHO
H3C
CH2NH2Cofactor (N donor)Pyridoxamine-PO4
Alanine-Pyruvate Aminotransferase
COO-
C=OCH2
COO-
CH2
+COO-
H3N-C-HCH3
+
N
CH2OPHO
H3C
CH
O
+H3N-C-H
COO-
CH2
COO-
CH2
+COO-
CH3
C=O
N
CH2OPHO
H3C
CH2NH2
N
CH2OPHO
H3C
CH
Oforward reverse
Alanine
Enz-CHO(E-B6-al)
Enz-NH2
(E-B6-am)
Pyruvate -Ketoglutarate
Enz-NH2 Enz-CHO
Glutamate
Ordered Ping-Pong Mechanism
Mechanism
In Out In Out
Glutamate Metabolism
COO-
C=OCH2
COO-
CH2
+ NAD(P)+
+ NH4+
+ H2O + NAD(P)H + H+
Glutamate dehydrogenase
Urea cycle
specific for glutamate
requires NAD+
Forward Reaction
delivers NH4+ to urea cycle
Reverse Reactionspecific for -ketoglutarate
requires NADPH
Fixes NH4+, prevents toxicity
H3N-C-HCOO-
CH2
COO-
CH2
+
Glutamine Metabolism
H3N-C-HCOO-
CH2
COO-
CH2
+
+ ADP + Pi
Glutamine Synthetase
H3N-C-HCOO-
CH2
COO-
CH2
+
Glutaminase
+ NH4+
H2O
H3N-C-HCOO-
CH2
CH2
+
C=OOPO3
=
+ ATP + NH4+
H3N-C-HCOO-
CH2
CH2
+
C=ONH2
L-glutamine
intermediateGlutamate-PO4
Urea
Overall Scheme Using Alanine as an Example
Alanine Pyruvate
-ketoglutarate glutamate
NH4+
Urea
Amino transferase with pyridoxal-5’-PO4
Glutamate dehydrogenase with NAD+
Glutaminase with H2O
glutamine
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
NH4+ + HCO3
- + 2 ATP
Synthesis of Carbamoyl-PO4
H2NC
O
O-P-OO
O~
High energy bond
+ 2 ADP + Pi
Carbamoyl phosphate Synthetase I
Ornithine
Citrulline
Argininosuccinate
Arginine
Carbamoyl-PO4
Aspartate
Urea
ATP
Urea Cycle
H2ONHCH2
CH2
CH2
COO-C
H3NH
H2N=CNH2+
NH3
CH2
CH2
CH2
COO-C
H3NH
+
CH2N NH2
O
COPO3H2N
O
COO-
CH2
H3N+-C-H
NH3
CH2
CH2
+
+
O=C
COO-
CH2
H3N+-C-H
NH
CH2
CH2
NH2
+ OPO3=
Ornithine
Carbamoyl-PO4 Citruline
Reactions of Urea Cycle
O=C
COO-
CH2
H3N+-C-H
NH
CH2
CH2
NH2
+
COO-
CH2
COO-
H-C-NH3
+
=C
COO-
CH2
H3N+-C-H
NH
CH2
CH2
NH2
COO-
CH2
COO-
H-C-NL-Aspartate
Argininosuccinate
ATP ADP + Pi
Mitochondria
Cytosol
=C
COO-
CH2
H3N+-C-H
NH
CH2
CH2
NH2
COO-
CH2
COO-
H-C-N =C
COO-
CH2
H3N+-C-H
NH
CH2
CH2
NH2
H2N+
COO-
COO-
CH2
C-OHH
+
COO-
COO-
C
C
H
H
COO-
COO-
CH2
C=O
COO-
CH2
COO-
H-C-NH3
+
Fumarate
L-MalateOxaloacetateL-Aspartate
Cytosol
L-Arginine
=C
COO-
CH2
H3N+-C-H
NH
CH2
CH2
NH2
H2N+
COO-
CH2
H3N+-C-H
NH3
CH2
CH2
+H2N
C
O
NH2
Urea
+
Ornithine
L-Arginine
H2O
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