1. PROTEIN METABOLISM: NITROGEN CYCLE; DIGESTION OF PROTEINS
Red meat is an important dietary source of protein nitrogen

2. The Nitrogen Cycle and Nitrogen Fixation
Nitrogen is needed for amino acids, nucleotides, etc
Atmospheric N2 is the ultimate source of biological nitrogen
Nitrogen fixation: biosynthetic process of the reduction of N2 to NH3 (ammonia)
Higher organisms are unable to fix nitrogen.
Some bacteria and archaea can fix nitrogen.

3. Nodules of Rhizobium bacteria
Symbiotic Rhizobium bacteria invade the roots of leguminous plants and form root nodules.
Nodules of Rhizobium bacteria
Rhizobium bacteria fix nitrogen supplying both the bacteria and the plants.
Archaea

4. The amount of N2 fixed by nitrogen-fixing microorganisms is about 60% of Earth's newly fixed nitrogen.
Lightning and ultraviolet radiation fix 15%
25% is fixed by industrial processes (fertilizer factories)

5. Nitrogen-fixing bacteria possess nitrogenase complex which can reduce N2 to ammonia
The nitrogenase complex consists of two proteins:
reductase, which provides electrons
nitrogenase, which uses these electrons to reduce N2 to NH3.
The transfer of electrons from the reductase to the nitrogenase is coupled to the hydrolysis of ATP.

6. Nitrogenase reaction:
The high-potential electrons come from protein ferredoxin, generated by photosynthesis or oxidative processes.
16 molecules of ATP are hydrolyzed for each molecule of N2 reduced.
Nitrogenase reaction:
N2 + 8 H+ + 8 e ATP 
2 NH3 + H ADP + 16 Pi
Reductase – dimer containing Fe-S clusters and ATP-binding site

7. The nitrogenase component is an 22 tetramer.
Contains P cluster (Fe-S) and FeMo cofactor.
FeMo cofactor is the site of nitrogen fixation.

8. NO2- and NO3- are used by higher plants
Ammonia in the presence of water becomes NH4+ which can be used by plants
NH4+ can be rapidly oxidized by soil bacteria Nitrosomonas and Nitrobacter to NO2- and NO3- (nitrification)
Nitrosomonas
NO2- and NO3- are used by higher plants
Another soil bacteria can reverse the nitrification process and convert NO2- and NO3- back to nitrogen
Nitrogen from plants and animals is recycled to soil (excretion of nitrogen in the form of urea or uric acid; decay of plants and animals) - nitrogen cycle

10. Assimilation of Ammonia
Ammonia generated from N2 is assimilated into amino acids such as glutamate or glutamine

11. A. Ammonia Is Incorporated into Glutamate
Reductive amination of a-ketoglutarate by glutamate dehydrogenase occurs in plants, animals and microorganisms
This reaction establishes the stereochemistry of the -carbon atom in glutamate. Only the L isomer of glutamate is synthesized.

12. B. Glutamine Is a Nitrogen Carrier
A second route in assimilation of ammonia is via glutamine synthetase
All organisms have both enzymes: glutamate dehydrogenase and glutamine synthetase.
Amino acids are used for the synthesis of proteins.
Animals and humans consume proteins.
Proteins undergo digestion in the stomach and intestine.

13. Protein digestion Digestion in Stomach
Stimulated by food acetylcholine, histamine and gastrin are released onto the cells of the stomach
The combination of acetylcholine, histamine and gastrin cause the release of the gastric juice.
Mucin - is always secreted in the stomach
HCl - pH (secreted by parietal cells)
Pepsinogen (a zymogen, secreted by the chief cells)
Hydrochloric acid:
Creates optimal pH for pepsin
Denaturates proteins
Kills most bacteria and other foreign cells

14. Pepsinogen (MW=40,000) is activated by the enzyme pepsin present already in the stomach and the stomach acid. 
Pepsinogen cleaved off to become the enzyme pepsin (MW=33,000) and a peptide fragment to be degraded.
Pepsin partially digests proteins by cleaving the peptide bond formed by aromatic amino acids: Phe, Tyr, Trp

15. Digestion in the Duodenum
Stimulated by food secretin and cholecystokinin regulate the secretion of bicarbonate and zymogens trypsinogen, chymotrypsinogen, proelastase and procarboxypeptidase by pancreas into the duodenum
Bicarbonate changes the pH to about 7
The intestinal cells secrete an enzyme called enteropeptidase that acts on trypsinogen cleaving it into trypsin

16. Trypsin converts chymotrypsinogen into chymotrypsin, procarboxypeptidase into carboxypeptidase and proelastase into elastase, and trypsinogen into more trypsin.
Trypsin which cleaves peptide bonds between basic amino acids Lys and Arg
Chymotrypsin cleaves the bonds between aromatic amino acids Phe, Tyr and Trp
Carboxypeptidase which cleaves one amino acid at a time from the carboxyl side
Aminopeptidase is secreted by the small intestine and cleaves off the N-terminal amino acids one at a time

17. Most proteins are completely digested to free amino acids
Amino acids and sometimes short oligopeptides are absorbed by the secondary active transport
Amino acids are transported via the blood to the cells of the body.

18. The ways of entry and using of amino acids in tissue
The sources of amino acids:
1) absorption in the intestine;
2) protein decomposition;
3) synthesis from the carbohydrates and lipids.
Using of amino acids:
1) for protein synthesis;
2) for synthesis of other nitrogen containing compounds (creatine, purines, choline, pyrimidine);
3) as the source of energy;
4) for the gluconeogenesis.

19. PROTEIN METABOLISM: PROTEIN TURNOVER; GENERAL WAYS OF AMINO ACIDS METABOLISM

20. PROTEIN TURNOVER
Protein turnover — the degradation and resynthesis of proteins
Half-lives of proteins – from several minutes to many years
Structural proteins – usually stable (lens protein crystallin lives during the whole life of the organism)
Regulatory proteins - short lived (altering the amounts of these proteins can rapidly change the rate of metabolic processes)
How can a cell distinguish proteins that are meant for degradation?

21. Ubiquitin - is the tag that marks proteins for destruction ("black spot" - the signal for death)
Ubiquitin - a small (8.5-kd) protein present in all eukaryotic cells
Structure:
extended carboxyl terminus (glycine) that is linked to other proteins;
lysine residues for linking additional ubiquitin molecules

22. Ubiquitin covalently binds to -amino group of lysine residue on a protein destined to be degraded.
Isopeptide bond is formed.

23. Mechanism of the binding of ubiquitin to target protein
E1 - ubiquitin-activating enzyme (attachment of ubiquitin to a sulfhydryl group of E1; ATP-driven reaction)
E2 - ubiquitin-conjugating enzyme (ubiquitin is shuttled to a sulfhydryl group of E2)
E3 - ubiquitin-protein ligase (transfer of ubiquitin from E2 to -amino group on the target protein)

24. Attachment of a single molecule of ubiquitin - weak signal for degradation.
Chains of ubiquitin are generated.
Linkage – between -amino group of lysine residue of one ubiquitin to the terminal carboxylate of another.
Chains of ubiquitin molecules are more effective in signaling degradation.

25. What determines ubiquitination of the protein?
1. The half-life of a protein is determined by its amino-terminal residue (N-terminal rule).
E3 enzymes are the readers of N-terminal residues.
2. Cyclin destruction boxes - specific amino acid sequences (proline, glutamic acid, serine, and threonine –PEST)

26. Digestion of the Ubiquitin-Tagged Proteins
What is the executioner of the protein death?
A large protease complex proteasome or the S proteasome digests the ubiquitinated proteins.
26S proteasome - ATP-driven multisubunit protease.
26S proteasome consists of two components:
20S - catalytic subunit
19S - regulatory subunit

27. 20S subunit
resembles a barrel
is constructed from 28 polipeptide chains which are arranged in four rings (two  and two )
active sites are located in  rings on the interior of the barrel
degrades proteins to peptides (seven-nine residues)

28. 19S subunit
made up of 20 polipeptide chains
controls the access to interior of 20S barrel
binds to both ends of the 20S proteasome core
binds to polyubiquitin chains and cleaves them off
possesses ATPase activity
unfold the substrate
induce conformational changes in the 20S proteasome (the substrate can be passed into the center of the complex)

29. GENERAL WAYS OF AMINO ACIDS METABOLISM
The fates of amino acids:
1) for protein synthesis;
2) for synthesis of other nitrogen containing compounds (creatine, purines, choline, pyrimidine);
3) as the source of energy;
4) for the gluconeogenesis.

30. Deamination of amino acids
The general ways of amino acids degradation:
Deamination
Transamination
Decarboxilation
The major site of amino acid degradation - the liver.
Deamination of amino acids
Deamination - elimination of amino group from amino acid with ammonia formation.
Four types of deamination:
- oxidative (the most important for higher animals),
- reduction,
- hydrolytic, and
- intramolecular

31. Reduction deamination: R-CH(NH2)-COOH + 2H+  R-CH2-COOH + NH3
amino acid fatty acid
Hydrolytic deamination:
R-CH(NH2)-COOH + H2O  R-CH(OH)-COOH + NH3
amino acid hydroxyacid
Intramolecular deamination:
R-CH(NH2)-COOH  R-CH-CH-COOH + NH3
amino acid unsaturated fatty acid

32. Oxidative deamination
L-Glutamate dehydrogenase plays a central role in amino acid deamination
In most organisms glutamate is the only amino acid that has active dehydrogenase
Present in both the cytosol and mitochondria of the liver

33. Transamination of amino acids
Transamination - transfer of an amino group from an -amino acid to an -keto acid (usually to -ketoglutarate)
Enzymes: aminotransferases (transaminases).
-amino acid
-keto acid

34. There are different transaminases
The most common:
alanine aminotransferase alanine + -ketoglutarate  pyruvate + glutamate
aspartate aminotransferase
aspartate + -ketoglutarate  oxaloacetate + glutamate
Aminotransferases funnel -amino groups from a variety of amino acids to -ketoglutarate with glutamate formation
Glutamate can be deaminated with NH4+ release

35. Mechanism of transamination
All aminotransferases require the prosthetic group pyridoxal phosphate (PLP), which is derived from pyridoxine (vitamin B6).
Ping-pong kinetic mechanism
First step: the amino group of amino acid is transferred to pyridoxal phosphate, forming pyridoxamine phosphate and releasing ketoacid.
Second step: -ketoglutarate reacts with pyridoxamine phosphate forming glutamate

36. Ping-pong kinetic mechanism of aspartate transaminase
aspartate + -ketoglutarate  oxaloacetate + glutamate

37. Enzyme: decarboxylases Coenzyme – pyrydoxalphosphate
Decarboxylation of amino acids
Decarboxylation – removal of carbon dioxide from amino acid with formation of amines.
amine
Usually amines have high physiological activity (hormones, neurotransmitters etc).
Enzyme: decarboxylases Coenzyme – pyrydoxalphosphate

38. Significance of amino acid decarboxylation
1. Formation of physiologically active compounds
GABA – mediator of nervous system
glutamate
gamma-aminobutyric acid (GABA)
histamine
histidine
Histamine – mediator of inflammation, allergic reaction.

39. 2. Catabolism of amino acids during the decay of proteins
Enzymes of microorganisms (in colon; dead organisms) decarboxylate amino acids with the formation of diamines.
ornithine
putrescine
lysine
cadaverine

40. PROTEIN METABOLISM: UTILIZATION OF AMMONIA IONS; UREA CYCLE
The basic features of nitrogen metabolism were elucidated initially in pigeons

41. AMMONIA METABOLISM The ways of ammonia formation
1. Oxidative deamination of amino acids
2. Deamination of physiologically active amines and nitrogenous bases.
3. Absorption of ammonia from intestine (degradation of proteins by intestinal microorganisms results in the ammonia formation).
4. Hydrolytic deamination of AMP in the brain (enzyme – adenosine deaminase)

42. Ammonia is a toxic substance to plants and animals (especially for brain)
Normal concentration: mol/l ( mg/l)
Ammonia must be removed from the organism
Terrestrial vertebrates synthesize urea (excreted by the kidneys) - ureotelic organisms
Birds, reptiles synthesize uric acid
Urea formation takes place in the liver

43. Peripheral Tissues Transport Nitrogen to the Liver
Two ways of nitrogen transport from peripheral tissues (muscle) to the liver:
Glutamate is not deaminated in peripheral tissues
1. Alanine cycle. Glutamate is formed by transamination reactions

44. Nitrogen is then transferred to pyruvate to form alanine, which is released into the blood.
The liver takes up the alanine and converts it back into pyruvate by transamination.
The glutamate formed in the liver is deaminated and ammonia is utilized in urea cycle.

45. 2. Nitrogen can be transported as glutamine.
Glutamine synthetase catalyzes the synthesis of glutamine from glutamate and NH4+ in an ATP-dependent reaction:

46. THE UREA CYCLE
Urea cycle - a cyclic pathway of urea synthesis first postulated by H.Krebs
The sources of nitrogen atoms in urea molecule:
aspartate;
NH4+.
Carbon atom comes from CO2.

47. The free ammonia is coupling with carbon dioxide to form carbamoyl phosphate
Two molecules of ATP are required
Reaction takes place in the matrix of liver mitochondria
Enzyme: carbamoyl phosphate synthetase (20 % of the protein of mitochondrial matrix)

48. Enzyme: ornithine carbamoyltransferase
Carbamoyl phosphate donates carbamoyl group to ornithine
The product - citruilline
Enzyme: ornithine carbamoyltransferase
Reaction takes place in the mitochondrial matrix
Citrulline leaves the matrix and passes to the cytosol

49. In the cytosol citrulline in the presence of ATP reacts with aspartate to form argininosuccinate
Enzyme: argininosuccinate synthetase

50. Argininosuccinate is cleaved to free arginine and fumarate
Enzyme: argininosuccinate lyase
The fumarate enters the tricarboxylic acid cycle

51. Arginine is hydrolyzed to generate urea and ornithine
Enzyme: arginase (present only in liver of ureotelic animals)
Ornithine is transported back into the mitochondrion to begin another cycle
Urea is excreted (about 40 g per day)

52. The urea cycle

53. The Linkage between Urea Cycle, Citric Acid Cycle and Transamination of Oxaloacetate
Fumarate formed in urea cycle enters citric acid cycle and is converted to oxaloacetate.
Fates of oxaloacetate:
transamination to aspartate,
conversion into glucose,
condensation with acetyl CoA to form citrate,
conversion into pyruvate.