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

2. 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 0.8-2.5 (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

3. 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

4. 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

5. 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

6. 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.

7. 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.

8. PROTEIN TURNOVER How can a cell distinguish proteins that are meant for degradation? 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)

9. 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.

10. 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

11. Reduction deamination: R-CH(NH 2 )-COOH + 2H +  R-CH 2 -COOH + NH 3 amino acid fatty acid Hydrolytic deamination: R-CH(NH 2 )-COOH + H 2 O  R-CH(OH)-COOH + NH 3 amino acid hydroxyacid Intramolecular deamination: R-CH(NH 2 )-COOH  R-CH-CH-COOH + NH 3 amino acid unsaturated fatty acid

12. 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

13. 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  -amino acid

14. 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 NH 4 + release

15. Mechanism of transamination All aminotransferases require the prosthetic group pyridoxal phosphate (PLP), which is derived from pyridoxine (vitamin B 6 ). 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 Ping-pong kinetic mechanism

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

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

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

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

20. 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)

21. Ammonia is a toxic substance to plants and animals (especially for brain) Normal concentration: 25-40  mol/l (0.4-0.7 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

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

23. 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.

24. 2. Nitrogen can be transported as glutamine. Glutamine synthetase catalyzes the synthesis of glutamine from glutamate and NH 4 + in an ATP- dependent reaction:

25. Urea cycle - a cyclic pathway of urea synthesis first postulated by H.Krebs THE UREA CYCLE The sources of nitrogen atoms in urea molecule: - aspartate; - NH 4 +. Carbon atom comes from CO 2.

26. 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)

27. 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

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

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

30. 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)

31. The urea cycle

32. 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: (1)transamination to aspartate, (2)conversion into glucose, (3)condensation with acetyl CoA to form citrate, (4)conversion into pyruvate.

33. SPECIFIC WAYS OF AMINO ACID CATABOLISM The carbon skeletons of 20 fundamental amino acids are funneled into seven molecules:  pyruvate,  acetyl CoA,  acetoacetyl CoA,   -ketoglutarate,  succinyl CoA,  fumarate,  oxaloacetate. After removing of amino group the carbon skeletons of amino acids are transformed into metabolic intermediates that can be converted into glucose, fatty acids, ketone bodies or oxidized by the citric acid cycle.

34. Fates of carbon skeleton of amino acids

35. Glucogenic vs ketogenic amino acids Glucogenic amino acids (are degraded to pyruvate or citric acid cycle intermediates) - can supply gluconeogenesis pathway Ketogenic amino acids (are degraded to acetyl CoA or acetoacetyl CoA) - can contribute to synthesis of fatty acids or ketone bodies Some amino acids are both glucogenic and ketogenic

36. Pyruvate as an Entry Point into Metabolism

37. Oxaloacetate as an Entry Point into Metabolism Aspartate and asparagine are converted into oxaloacetate aspartate +  -ketoglutarate  oxaloacetate + glutamate Asparagine is hydrolyzed to NH 4 + and aspartate, which is then transaminated.

38.  -Ketoglutarate as an Entry Point into Metabolism

39. Succinyl Coenzyme A Is a Point of Entry for Several Nonpolar Amino Acids

40. Methionine Degradation S-adenosylmethionine (SAM) - a common methyl donor in the cell

41. Homocysteine (< 15 μmol/L) Hyperhomocysteinemia can results in: Vascular diseases, endothelial dysfunction, atherosclerosis, thrombophilia Skeletal anomalies retardation of mental development Ectopic lens Alzheimer's disease Kidneys insufficiency Colorectal cancer Homocysteine

42. The Conversion of Branched-Chain Amino Acids The degradative pathways of valine and isoleucine resemble that of leucine. Isoleucine yields acetyl CoA and propionyl CoA Valine yields CO 2 and propionyl CoA. branched- chain dehydrogen ase

43. Degradation of Aromatic Amino Acids Acetoacetate, fumarate, and pyruvate — are common intermediates. Molecular oxygen is used to break an aromatic ring. homogenti sate oxidase +O 2 tetrahy dro- biopteri n PA hydro- xylase

44. Tryptophan degradation requires several oxygenases Pyruv ate

45. INBORN ERRORS OF AMINO ACIDS METABOLISM Alcaptonuria - inherited disorder of the tyrosine metabolism caused by the absence of homogentisate oxidase.  homogentisic acid is accumulated and excreted in the urine  turns a black color upon exposure to air  In children:  urine in diaper may darken  In adults:  darkening of the ear  dark spots on the on the sclera and cornea  arthritis

47. Urine turns a black color upon exposure to air Alcaptonuria Aortic valve stenosis in alcaptonuria Accumulation of oxidized homogentisic acid pigment in connective tissue (ochronosis) Arthritis of the spine is a complication of alkaptonuria ochronosis

48. Phenylketonuria is caused by an absence or deficiency of phenylalanine hydroxylase or of its tetrahydrobiopterin cofactor. Phenylalanine accumulates in all body fluids and converts to phenylpyruvate.  Defect in myelination of nerves  The brain weight is below normal.  Mental and physical retardations.  The life expectancy is drastically shortened. Diagnostic criteria:  phenylalanine level in the blood  FeCl 3 test  DNA probes (prenatal)

49. Albinism – genetically determined lack or deficit of enzyme tyrosinase Tyrosinase in melanocytes oxidases tyrosine to DOPA and DOPA-chinone tyrosinase Phenylalanine Tyrosine Tyroxine Melanin DOPA Dopamine Norepinephrine Epinephrine

50. Symptoms of albinism: inhibition of production or lack of melanin in skin, hair, eyes increased sensitivity to sunlight increased risk of skin cancer development sun burns photophobia decrease of vision acuity strabismus, nystagmus

51. Maple syrup urine disease - the disorder of the oxidative decarboxylation of  -ketoacids derived from valine, isoleucine, and leucine caused by the missing or defect of branched-chain dehydrogenase. The levels of branched-chain amino acids and corresponding  -ketoacids are markedly elevated in both blood and urine. The urine has the odor of maple syrup The early symptoms:  lethargy  ketoacidosis  unrecognized disease leads to seizures, coma, and death  mental and physical retardation

52. Nitric oxide (. N=O) is a gas which can diffuse rapidly into cells, and is a messenger that activates guanylyl cyclase (GMP synthesis) NO relaxes blood vessels, lowers blood pressure, and is a neurotransmitter in the brain SYNTHESIS OF NITRIC OXIDE (NO) FROM ARGININE

53. Nitroglycerin is converted to NO and dilates coronary arteries in treating angina pectoris

54. Conversion of arginine to NO via nitric oxide synthase

55. SPECIFIC WAYS OF AMINO ACID SYNTHESIS Plants and microorganisms can make all 20 amino acids Humans can make only 11 of the 20 amino acids (“nonessential” amino acids) Nonessential amino acids for mammals are usually derived from intermediates of glycolysis or the citric acid cycle The others are classed as "essential" amino acids and must be obtained in the diet

56. A deficiency of even one amino acid results in a negative nitrogen balance. In this state, more protein is degraded than is synthesized.

57. The pathways for the biosynthesis of amino acids are diverse Common feature: carbon skeletons come from intermediates of  glycolysis,  pentose phosphate pathway,  citric acid cycle. All amino acids are grouped into families according to the intermediates that they are made from