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1. Amino Acids and proteins By Alemu Adela DBU 2018

2. Objectives Describe the structures of amino acids List the biological importance of amino acids and proteins Understand the basis of amino acid classification List the properties of amino acids Describe the structural classification of proteins Know the structure and functions of heme proteins( Hemoglobin and myoglobin )

3. Amino Acids Amino Acids are the building and functional units of proteins. 300 amino acids occur in nature. Only 20 of them occur in proteins. Structure of amino acids: Each amino acid has 4 different groups attached to the same tetrahedral carbon atom . : Amino group, COOH gp , Hydrogen atom and Side Chain d istinct R-groups , that distinguish one amino acid from another, R N.B. Proline * has cyclic structure other than the common amino acid structure( imino acid)

4. Biomedical Importance They are polymerized to form proteins . the L-α-amino acids and their derivatives functions as :- Source of energy, nerve transmission and the biosynthesis of carbohydrates, porphyrins , purines, pyrimidines and urea . Short polymers of amino acids called peptides perform prominent roles in the:- Neuroendocrine system as hormones , hormone-releasing factors, neurotransmitters. Detoxification of free radicals(GSH) 3/14/2023 4

5. Classification of amino acids I- Chemical classification: is based on the R group of the AA According to net charge on amino acid, R Classified in to three: Neutral amino acids Basic amino acids Acidic amino acids

6. A- Neutral amino acids includes 1- Glycine R= H 2- Alanine R= CH 3 3- Branched chain amino acids: R is branched such as in: a- Valine R= isopropyl b- Leucine R= isobutyl c- Isoleucine R = isobutyl 4- Sulfur containing amino acids: Cysteine & Methionine 5- hydroxy amino acids: Serine and Threonine 6- aromatic amino acids: - Phenyl alanine , Tyrosine & Tryptophan 7- Amides: Aspargine and Glutamine 8- Proline ( imino acids ) : In proline , amino group enters in the ring formation being α - imino gp

7. B- Basic amino acids: Contain two or more NH 2 groups or nitrogen atoms that act as a base At physiological pH, basic amino acids will be positively charged . Lysine, Arginine (contains guanido group) and Histidine are basic AAs

8. C- Acidic Amino acids: At physiological pH will carry negative charge Aspartic acid ( aspartate ) and Glutamic acid (glutamate)are acidic amino acids

9. According to polarity of side chain (R): A- Polar amino acids: R contains polar hydrophilic group so can forms hydrogen bond with H 2 O and are predominantly found on the exterior surfaces of proteins in aqueous solution In those amino acids, R may contain: 1- OH group 2- SH group 4- NH 2 group 5- COOH group B- Non polar amino acids: R is alkyl hydrophobic group which can’t enter in hydrogen bond formation and reside predominantly in the interior of proteins

10. II- Nutritional classification: 1- Essential amino acids: These amino acids can’t be formed in the body and so, it is essential to be taken in diet. Their deficiency affects growth, health and protein synthesis. 2- Semiessential amino acids: These are formed in the body but not in sufficient amount for body requirements especially in children. Summary of essential and semiessential amino acids: Villa HM = Ten Thousands Pound V= valine i = isoleucine l= lysine l= leucine A = arginine * H= histidine * M= methionine T= tryptophan Th = threonine P= phenyl alanine * * = phenyl alanine , arginine and histidine are semiessential 3- Non essential amino acids: These are the rest of amino acids that are formed in the body in amount enough for adults and children. They are the remaining 10 amino acids.

11. III- Metabolic classification: According to metabolic or degradation products of amino acids they can be: 1- Ketogenic amino acids: which give ketone bodies . Lysine and Leucine are the only pure ketogenic amino acids. 2- Mixed ketogenic and glucogenic amino acids: which give both ketonbodies and glucose.These are: isoleucine , phenyl alanine , tyrosine and tryptophan. 3- Glucogenic amino acids: Which give glucose. They include the rest of amino acids. These amino acids by catabolism yields products that enter in glycogen and glucose formation.

12. Amphoteric properties : have both basic and acidic groups and so can act as base or acid. Neutral amino acids exist in aqueous solution as “ Zwitter ion” i.e. contain both positive and negative charge. Thus, amino acids can act as buffers. Chemical properties: 1- Reactions due to COOH group: Salt formation with alkalis, Ester formation with alcohols, Amide formation with amines Decarboxylation 2- Reactions due to NH2 group: Deamination and reaction with ninhydrin reagent. Ninhydrin reagent reacts with amino group of amino acid yielding blue colored product. The intensity of blue color indicates quantity of amino acids present. Properties of amino acids

13. Peptides The 20 amino acids are linked together through “ peptide bond forming peptides and proteins (what’s the difference?). The chains containing less than 50 amino acids are called “peptides”, while those containing greater than 50 amino acids are called “proteins”. Peptide bond formation: α -carboxyl group of one amino acid forms a covalent peptide bond with α -amino group of another amino acid by removal of a molecule of water. The result is : Dipeptide By the same way, the dipeptide can then forms a second peptide bond with a third amino acid to give Tripeptide . Repetition of this process generates a polypeptide or protein of specific amino acid sequence.

14. Peptide bond formation: - Each polypeptide chain starts on the left side by free amino group of the first amino acid enter in chain formation . It is termed N- terminus - Each polypeptide chain ends on the right side by free COOH group of the last amino acid and termed C-terminus

15. Glutathione is a tripeptide formed from 3 AAs:  - glu , cys and gly . It is formed in liver cytoplasm by the help of ATP and  -glutamyl-cysteine synthetase and glutathione synthetase . Glutathione exists in 2 states: reduced (GSH); and oxidized (GSSH). GSH is involved in a number of reduction reactions that helps protect cells against damage from reactive oxygen species (ROS). Regeneration of GSH from GSSH is catalyzed by glutathione reductase, which uses NADPH as a reductant. It is also important for amino acid intestinal absorption and transportation, and is used as a Coenzyme for a number of enzymes 19 GSSH Biologically important peptide glutathione

16. Proteins Proteins are physically and functionally complex macromolecules. Perform multiple critically important roles. Maintains cell shape and integrity : cytoskeleton Form the contractile machinery of muscle: Actin and myosin Protects from foreign invaders : antibodies Catalyze reactions: Enzymes Enable cells to sense and respond : Receptors Transport small molecules and ions: hemoglobin These functions and others arise from specific binding interactions and conformational changes in the structure of a properly folded proteins

17. The linear sequence of amino acids ( primary structure ) folds into helices or sheets ( secondary structure) Which pack into a globular or fibrous domain (tertiary structure). Some individual proteins self-associate into complexes (quaternary structure) complexes can consist of tens to hundreds of subunits ( supramolecular assemblies) Protein structure

18. Primary structure The linear sequence of amino acids This structural level stems from amino acids chemically bound through peptide bonds Determine the three dimensional structure of proteins A knowledge of the primary structure of proteins is essential in order to diagnose genetic diseases Abnormal sequences of amino acids typically lead to improper folding in the resulting protein, which can then produce a loss of its function

19. Secondary structure The local structure of the polypeptide chain Determined by hydrogen bond interactions between the carbonyl oxygen group of one peptide bond and the amide hydrogen of another nearby peptide bond. Two types α-helix The β-pleated sheet

20. α-helix The α-helix is a rod-like structure with the peptide chain tightly coiled and the side chains of amino acid residues extending outward from the axis of the spiral. Intrachain hydrogen bonds maintain the coiled structure Each turn of the helix contains 3.6 amino acids Proline disrupts an -helix (kink)

21. The β- pleated sheet An extended structure as opposed to the coiled α-helix All of the peptide bond moieties involved in hydrogen bonding to create a pleated sheet structure It is pleated because the carbon-carbon (C-C) bonds are tetrahedral and cannot exist in a planar configuration It can be parallel or anti parallel depend on the direction of the poly peptide chain that forms it

22. A β -pleated sheet.

23. Tertiary structure Three-dimensional, folded and biologically active conformation of a protein This structure reflects the overall shape of the molecule Stabilized by Covalent disulfide bonds Hydrogen bonds Salt bridges(ionic interaction) Hydrophobic interactions

24. Domains and folds Domains and. A section of protein structure sufficient to perform a particular chemical or physical task such as binding of a substrate or other ligand . Folds: Within a domain, a combination of secondary structural elements forms a fold, such as the nucleotide binding fold, or an actin fold. Folds are defined by their similarity in a number of different proteins.

25. V. Quaternary structure of proteins Is the arrangement (spatial relationships ) of polypeptide subunits If two subunits – dimeric If three subunits – trimeric If several subunits – multimeric Subunits held together by intermolecular forces (e.g. hydrogen bonds, ionic bonds, hydrophobic interactions) Subunits may either operate independently of each other, or may work cooperatively (e.g. hemoglobin)

26. Denaturation of proteins Occurs when the protein’s secondary and tertiary structures are disrupted Does not lead to hydrolysis of peptide bonds Denaturing agents include heat , organic solvents , mechanical mixing , strong acids or bases , detergents , and lead and mercury ions Most proteins remain permanently disordered once denatured and become insoluble Denaturation may be reversible (under ideal conditions)

27. Classification Proteins can be classified as globular and fibrous Globular proteins Compact and roughly spherical have axial ratios(the ratio of their shortest to longest dimensions) of not over 3 Most enzymes and heme proteins are globular proteins Fibrous proteins Adopt highly extended conformations. Possess axial ratios of 10 or more Many structural proteins .

28. Hemoglobin and myoglobin Globular hemeproteins important for oxygen binding The proteins by themselves cannot bind oxygen. They employ heme , the porphyrin ring, for the purpose . Has a very similar primary structure Myoglobin is a single polypeptide that has one O2 binding site Hemoglobin is a tetramer composed of two different types of subunits with 4 O2 binding site A comparison between myoglobin and hemoglobin illustrates the advantages of a multisubunit quaternary structure.

29. 33 Myoglobin 153 AA, single polypeptide with tertiary structure →water soluble Found mainly in muscle tissue where it serves as intracellular reservoir of oxygen. Binds very tightly with oxygen and releases it only at very low partial pressure of oxygen i.e. at the time of extreme oxygen deprivation in muscles . The released oxygen can then be utilized for the metabolic activity of muscle cells.

30. Conti…. 80% made up of α-helical secondary structure with 8 separate right handed regions, designated A -H Interior of the molecule is largely hydrophobic , non-polar AAs Surface of the molecule carries the polar/ionic AAs Primarily stabilized by non-covalent forces but no disulfide bonds Carries a heme prosthetic group inserted into a hydrophobic pocket Each heme is a protoporphyrin -IX moiety and contains one central coordinately bound iron atom Iron is normally in the ferrous (Fe 2+ ) form and serves as the oxygen binding site

31. 35 Physiological importance of Myoglobin Myoglobin in tissues (especially in muscle) accepts oxygen from Hb in circulating arterial blood The p50 (oxygen partial pressure required for half saturation) for myoglobin is very low At the oxygen concentration existing in tissues, myoglobin is nearly saturated only in rapidly exercising muscle or conditions of hypoxia (extreme oxygen deprivation) would myoglobin give away its oxygen Due to its hyperbolic oxygen dissociation curve and very low p50 , myoglobin is unsuitable for the transport of oxygen

32. hemoglobin Hemoglobins are tetramers Consists of four globin subunit and four heme prosthetic group found in erythrocytes Heme prosthetic group binds oxygen The subunit composition of the principal hemoglobins are α2 β2 ( HbA ; normal adult hemoglobin), α2 γ2( HbF ; fetal hemoglobin), α2 S2( HbS ; sickle cell hemoglobin), Responsible for binding oxygen in the lung and transporting the bound oxygen throughout the body The quaternary structure of hemoglobin leads to physiologically important cooperative interactions between the subunits

33. Structure of hemoglobin

34. O2 binds to HGB cooperatively When oxygen binds to the first subunit of deoxyhemoglobin it increases the affinity of the remaining subunits for oxygen As additional oxygen is bound to the second and third subunits, oxygen binding is further, incrementally, strengthened, so that at the oxygen tension in lung alveoli, hemoglobin is fully saturated with oxygen As oxyhemoglobin circulates to deoxygenated tissue, oxygen is incrementally unloaded(removed) and the affinity of hemoglobin for oxygen is reduced. Thus at the lowest oxygen tensions found in very active tissues the binding affinity of hemoglobin for oxygen is very low allowing maximal delivery of oxygen to the tissue.  This phenomenon of tetrameric proteins are termed as Cooperative interactions , and have critical importance in aerobic life

35. In other words The binding of the first oxygen to HGB causes Profound changes in the quaternary structure of HGB accompany the high-affinity O2 induced transition of hemoglobin from the low-affinity T (taut) state to the high-affinity R (relaxed) state. These changes significantly increase the affinity of the remaining unoxygenated hemes for O2 as subsequent binding events require the rupture of fewer salt bridges Result in easier access of oxygen to the iron atom of the second heme and thus a greater affinity of the hemoglobin molecule for a second oxygen molecule.  The low affinity HG is known as the taut (T) state The high affinity HGB is known as relaxed(R) state

36. Hemoglobin saturation: Is the % of hemoglobin molecules carrying oxygen depends on PO2 of the blood: When PO2 is high, as in pulmonary capillaries, O2 binds with the Hb when PO2 is low, as in tissue capillaries, O2 is released from the Hb

37. P 50 of Hb The quantity P50 is the partial pressure of O2 that half-saturates a given hemoglobin. If the P50 is low, Hb has a higher affinity for O2 For example, values of P50 for HbA and HbF are 26 and 20 mm Hg, respectively. In the placenta, this difference enables HbF to extract oxygen from the HbA in the mother’s blood.

38. transport of CO 2  from the tissues to the blood with delivery of O 2  to the tissues.

39. CO 2  and O 2 Transport by Hb Hemoglobin transports 15% of CO2 and protons from peripheral tissues to the lungs. Much of the remaining CO2 is carried as bicarbonate, which is formed in erythrocytes by the hydration of CO2 to carbonic acid (H2CO3) →by carbonicanhydrase . At the pH of venous blood, H2CO3 dissociates into bicarbonate and a proton →lower pH of peripheral tissues, stabilizes the T state and thus enhances the delivery of O2.

40. In the lungs O2 binds to deoxyhemoglobin protons are released and combine with bicarbonate to form carbonic acid. Dehydration of H2CO3 catalyzed by carbonic anhydrase forms CO2, which is exhaled. This phenomena known as the B ohr effect A low Po 2 in peripheral tissues promotes the synthesis of 2,3-BPG in erythrocytes BPG therefore stabilizes deoxygenated (T state) hemoglobin by forming additional salt bridges

41. Regulation of oxygen binding  CO 2 , H + , and Cl –  exert negative effects on hemoglobin binding O 2 Increased 2,3-BPG concentration favors conversion of R form Hb to T form Hb and increases oxygen delivery by Hb to the tissue.

42. 47 Carbon Monoxide poisoning Carbon monoxide concentration is high in smoke from fire/ vehicular exhaust / cigarette . Oxygen and CO compete with each other for Hb binding CO binds with affinity 200 times that of oxygen , CO concentration of 1/200 that of oxygen can saturate 50% of Hb .

43. Carbon Monoxide poisoning When 30-50% of Hb is saturated with CO Throbbing headache Confusion Fainting When the saturation reaches 80% → rapidly fatal Oxygen, at high concentration, displaces CO from hemoglobin → CO poisoning can be overcome by elevated pO 2 Treatment → O 2 resuscitation

44. Hemoglobinopathies 1. Sickle cell anemia ( HbS ) Caused by a mutation  glutamic acid is substituted by valine at AA 6. Results in hemoglobin tetramers that aggregate into arrays upon deoxygenation in the tissues. This aggregation leads to deformation of the red blood cell making it relatively inflexible and unable to traverse the capillary beds. At the end it leads to clogging of the fine capillaries.

45. 50 Hemoglobinopathies Sickle Cells and malaria Individuals heterozygous for sickle-cell Hb have a higher resistance to malaria Plasmodium falciparam spends a portion of its life cycle in RBCs The increased fragility of the sickled cells tends to interrupt this cycle . Malarial parasite decreases the pH in RBCs and the low pH increases the sickling of the cells. The parasite into the cells promotes sickling , followed by lysis . Sickled cells have low concentrations of K + , which is highly unsuitable for the parasite to survive.

46. Hemoglobinopathies 2. Thalassemias Are the result of abnormalities in hemoglobin synthesis Deficiencies in β- globin synthesis result in the  β- thalassemias  which causes premature red cell destruction in the bone marrow and spleen. Deficiencies in α- globin synthesis result in the  α- thalassemias which leads to reduced oxygen carrying capacity.