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Nitrogen Metabolism

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

brittney-foley
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Nitrogen Metabolism

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  1. Nitrogen Metabolism • Protein degradation and turnover • Amino acid degradation and urea cycle • Nitrogen cycle • Nitrogen fixation • Amino acid biosynthesis • Amino acid derivatives

  2. 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

  3. 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.)

  4. dC Rate of Turnover = = KS - KDC dt 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 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

  5. dC = 0 dt Steady-state is achieved when the amount of protein synthesized per unit time equals the amount being destroyed 0.693 KDC = KS t 1/2 = KD C Protein concentration (enzyme activity) Stop protein synthesis, measure rate of decay Hours after stopping synthesis

  6. Steps in Protein Degradation ATP AMP + PPi Transformation to a degradable form (Metal oxidized, Ubiquination, N-terminal residues, PEST sequences) Lysosomal Digestion 26S Proteasome digestion 7  type, 7  type subunits Proteolysis to peptides KFERQ 8 residue fragments Ubiquination N-end rule: DRLKF: 2-3 min AGMSV: > 20 hr PEST: Rapid degradation

  7. Glycine at C terminal of Ubiquitin Ubiquitin COO- Ubiquitin activating enzyme ATP E1 HS Ubiquitin conjugating enzyme 20 or more per cell AMP + PPi O C S E1 3 E2 SH NH3+ N HS E2 HS E1 NH3+ N H3N+ O 3 C S E2 N O O O O ATP C C C C NH Ubiquitin- specific proteases (26S proteasome) E3 AMP + PPi Poly Ubiquitin Degraded protein + Ubiquitin Activation of Ubiquitin Ubiquitin ligase Ubiquination Page 1075

  8. 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

  9. 19S 20S Catalysis in beta 19S 7 alpha 7 beta Subunits 26S Proteasome (2000 kD) Opening for ubiquinated protein to enter 8-residue peptides diffuse out

  10. Amino Acids Amine Group Carbon Skeleton Glutamate Biosynthesis Degradation Amino Acids CO2 + H2O Urea Amino Acid Derivatives

  11. COO- Amine group acceptor COO- + H3N-C-H C=O CH2 CH2 CH2 CH2 COO- Amine group donor COO- -Ketoglutarate-Glutamate -Kg L-glutamate AA1 + -KG -ketoacid + glutamate acceptor donor Amino transferases Requires pyridoxal-5’-phosphate

  12. CH2OH CH2NH2 O HO HO C CH2OH CH2OP H HO CH2OP H3C H3C N N H3C N Vitamin B6 Pyridoxine Cofactor (N acceptor) Pyridoxal-5’-PO4 Cofactor (N donor) Pyridoxamine-PO4

  13. COO- COO- COO- + H3N-C-H + COO- C=O H3N-C-H CH2 C=O CH2 CH3 CH2NH2 CH2 CH3 O O CH2 HO COO- C C CH2OP COO- H H HO HO CH2OP CH2OP H3C N H3C H3C N N Alanine-Pyruvate Aminotransferase + + forward reverse

  14. Mechanism Alanine Glutamate -Ketoglutarate Pyruvate In Out In Out Enz-CHO (E-B6-al) Enz-NH2 (E-B6-am) Enz-NH2 Enz-CHO Ordered Ping-Pong Mechanism

  15. COO- COO- + H3N-C-H C=O CH2 CH2 CH2 CH2 COO- COO- Forward Reaction Reverse Reaction Glutamate Metabolism + NAD(P)+ + H2O + NAD(P)H + H+ Glutamate dehydrogenase + NH4+ Urea cycle specific for glutamate specific for -ketoglutarate requires NAD+ requires NADPH delivers NH4+ to urea cycle Fixes NH4+, prevents toxicity

  16. COO- COO- COO- COO- + + + + H3N-C-H H3N-C-H H3N-C-H H3N-C-H CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 COO- COO- C=O C=O NH2 OPO3= Glutamate-PO4 intermediate Urea Glutamine Metabolism + ATP + NH4+ + ADP + Pi Glutamine Synthetase L-glutamine H2O Glutaminase + NH4+

  17. glutamine Overall Scheme Using Alanine as an Example Amino transferase with pyridoxal-5’-PO4 Alanine Pyruvate -ketoglutarate glutamate Glutamate dehydrogenase with NAD+ Glutaminase with H2O NH4+ Urea Glutamate and glutamine are the only donors of NH3 to the Urea Cycle

  18. 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

  19. O O ~ O-P-O H2N C O High energy bond Synthesis of Carbamoyl-PO4 NH4+ + HCO3- + 2 ATP + 2 ADP + Pi Carbamoyl phosphate Synthetase I

  20. NH2 + + NH NH3 H2N=C CH2 CH2 CH2 CH2 CH2 CH2 H H O C C COO- COO- H3N H3N NH2 H2N Citrulline Aspartate Carbamoyl-PO4 ATP Urea Cycle Ornithine Argininosuccinate Arginine H2O C Urea

  21. Reactions of Urea Cycle O COO- C COO- H2N OPO3 H3N+-C-H H3N+-C-H CH2 CH2 + + OPO3= CH2 CH2 CH2 Carbamoyl-PO4 CH2 NH Citruline + NH3 O=C Ornithine NH2 COO- COO- H3N+-C-H H3N+-C-H COO- ATP ADP + Pi CH2 CH2 CH2 + H-C-NH3 + CH2 CH2 CH2 CH2 COO- COO- NH NH CH2 L-Aspartate O=C =C H-C-N NH2 NH2 Argininosuccinate COO- Cytosol Mitochondria

  22. COO- C H H C H2N+ COO- COO- COO- H3N+-C-H H3N+-C-H COO- COO- COO- CH2 CH2 CH2 CH2 CH2 + H-C-NH3 CH2 CH2 H C=O C-OH CH2 CH2 COO- COO- COO- COO- NH NH CH2 =C =C H-C-N NH2 NH2 COO- Cytosol + Fumarate L-Arginine L-Malate L-Aspartate Oxaloacetate

  23. O C H2N NH2 H2N+ COO- H3N+-C-H CH2 CH2 CH2 NH =C NH2 COO- H3N+-C-H CH2 H2O CH2 + CH2 NH3 + Urea Ornithine L-Arginine Return to Mitochondria

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