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SFA 2073 Topic II Amino Acid & Proteins

SFA 2073 Topic II Amino Acid & Proteins

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SFA 2073 Topic II Amino Acid & Proteins

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  1. SFA 2073 Topic II Amino Acid & Proteins Nik Norma Nik Mahmood (PhD) Faculty Science & Technology Uni.Science Islam Malaysia NILAI, N.Sembilan

  2. OBJECTIVES • To Classify amino acids according to their structures and properties. • To explain the meaning of pKa and pI of amino acids • To understand the biochemical benefit of amino acids • To describe the importance of some amino acids in the synthesis of important compounds • To understand the biochemical benefit of proteins • To Classify proteins according to their structures and properties. • Relate the structure of proteins to their functions using specific examples. • To understand the importance of amino acids & protein in biochemical efficiency.

  3. Discussion Order: • Structure & Function Of: - Amino Acid - Proteins • Proteins : - digestion and absorption - metabolism - metabolic disorder disease • Amino Acid : - absorption and metabolism - metabolic disorder disease

  4. INTRODUCTION: Expression of Concentration (the various expressions of concentrations used). At the end of this lecture, students should be able to: • Differentiate molarity and molality • Apply the units of concentration used in medicine (g%, mmol, g/dl, IU/I etc.) • Explain dilution, concentrated, saturated and supersaturated solution • Explain biological solution concentration ie hypertonic, hypotonic and isotonic.

  5. moles of solute (mol) Volume of solution (dm3 or liter) moles of solute (mol) m = mass of solvent (kg) M = Solution • I. There are several way to represent concentration of solution: a) Molarity (M) the number of moles of solute per liter solution. Unit: or or molar (M) b) Molality (m) the number of moles of solute per kg of solvent. Unit: molal (m) or molkg-1 moldm-3 molL-1

  6. 4.5

  7. II. Units of concentration used in biological science: a) Percent Composition by Mass (%) Ratio of the mass of solute to the mass of solution multiply by 100. eg 20g NaCl in 100 g salt solution 20 x 100 = 20 % sodium chloride solution 100 b) mmol: millimol = 1X 10-3 mol or 1 mol= 103 millimol c) g/dl : gdl-1 = g in 1 deciliter solution 10 dl = 1 L  1 dl = 10-1 L

  8. d) IU/I : International Unitis a unit of measurement for the amount of a substance, based on measured biological activity or effect. The unit is used for vitamins, hormones, some medications, vaccines, blood products, and similar biologically active substances. IU is not part of the International System of Units used in physics and chemistry. IU should not beconfused with the enzyme unit, also known as the International unit of enzyme activity and abbreviated as U. Mass equivalents of 1 IU • Insulin: 1 IU is the biological equivalent of about 45.5 μg pure crystalline insulin (1/22 mg exactly) • Vitamin A: 1 IU is the biological equivalent of 0.3 μg retinol, or of 0.6 μg beta-carotene • Vitamin C: 1 IU is 50 μg L-ascorbic acid • Vitamin D: 1 IU is the biological equivalent of 0.025 μg cholecalciferol/ergocalciferol • Vitamin E: 1 IU is the biological equivalent of about 0.667 mg d-alpha-tocopherol (2/3 mg exactly), or of 1 mg of dl-alpha-tocopherol acetate

  9. III. a) Making Dilutionsprocess of adding more solvent to a known solution. The moles of solute stay the same, moles = M x L In solution:initial Mole of solute = final Mole of soluteM1 V1 = M2 V2

  10. III. b) concentrated solution has less amount of water and more amount of the substance. For example concentrated H2SO4 has 2% water and 98% H2SO4 and dilute has less amount of substance and more amount of water c) saturated solution contains the maximum amount of a solute that will dissolve in a given solvent at a specific temperature. d) supersaturated solution contains more solute than is present in a saturated solution at a specific temperature. e) biological solution: concentration is described as hypertonicor hypotonic Hypertonic solution contain a high concentration of solute relative to another solutionon the other side of the membrane. Water from the other side will flow to this solution.

  11. solution

  12. Few notes for weak acid: • pH is a direct measure of the H+ concentration. • Ka: is acid dissociation/extent of ionisation constant, acidity constant. • pKa:The negative logarithm of Ka • pKb:The negative logarithm of the base protonation constant Kb • the extent of ionization of a weak acid (the pKa) influences the final concentration of H+ ions (the pH) of the solution. For a weak acid there is a relationship between pH and its pKa. This relationship is given by the Henderson–Hasselbalch equation: • pKa = pH + log [HA] / [A-] OR pH= pKa + log [A-] / [HA] can be CH3CO2- CH3 CZRCO2-

  13. Derivation of Henderson–Hasselbalch equation Ka = [H3O+] [CH3COO-] [CH3COOH] [H3O+] = Ka [CH3COOH] [CH3COO-] • X each by (- log) …… – log [H3O+] = – log Ka – log[CH3COOH] [CH3COO-] pH = pKa – log[CH3COOH] [CH3COO-] pH = pKa + log[CH3COO-] [CH3COOH] Henderson–Hasselbalch equation.

  14. Determination of pKa Titration of 100 mL 0.1 M CH3CO2H with 50 mL 0.1M NaOH CH3CO2H + NaOH CH3CO2‾+Na +H2O stoichiometric coefficient 1:1 Initial mole CH3CO2H = 0.01 Final mole CH3CO2ˉ =0.005 Unreacted CH3CO2H = 0.01- 0.005 = 0.005 pH value can be determined by using pH meter Substituting all the values in the equation, can get pKa By varying the volume of 0.1M NaOH in each titration can get the corresponding pH and pKa values

  15. Relationship between amino acids and protein: • Amino acids are building units of protein Protein n Peptide bonds Different coloured balls & box => Amino Acids

  16. Amino Acid: Structure & Function COOH • Amino acid (a.a) 20 altogether= std aa • all aa share a general formulaR-CH-NH2 • 1 aa differ from the other by the feature of –R • Classified based on : i) structure ii) side chain • aliphatic aa • non-polar • dicarboxylic aa • uncharge or non- ionic polar • diamino aa • charge or ionic • aromatic aa polar • heterocyclic aa

  17. Aliphatic Non-polar Amino Acid hydrophobicity Properties: - glycine and alanine are also found in the free form.

  18. Aromatic Amino Acid • Properties: tryptophane phenylalanine • Are non polar - absorb ultraviolet light (to different degree) - tyrosine has ionizable side chain

  19. Basic Amino Acid Histidine lysine arginine • Properties: • Are polar • - Are positively charged at pH values below their pKa’s • Are very hydrophilic • imidazole of histidine, at pH 7 exist predominantly in the neutral form.

  20. Acidic Amino Acid • Properties: • are polar • are negatively charge at physiological pH - the –COOH of side chain can form amide with an amino group.

  21. - iii) Nutritional Requirement • essential aa (8/9). Cannot be synthesized by the body • non-essential aa (12/11). Can be synthesized by the body * Essential in certaincases. Eg arginine & histidine are growth promoting factor there fore become essential in growing children

  22. - Amino acid is a derivative of organic (weak) acid. - Has 2 functional groups, carboxylic group (-COOH) and amino group (-NH2). Carboxylic (-COOH) and amine (-NH2)groups are capable of ionization: ―COOH ―COO‾ + H+ (2< pKa1< 2.5) ―N+H3 ―NH2 + H+ (9< pKa2< 9.5) ( ―N+H3 is a weaker acid ) - All aa is affected by pH: The net charge on the molecule in solution is affected by pH of their surrounding and can become more positively or negatively charged due to gain or the loss of protons (H+) respectively. eg. At pH~2.0 the amino group will be as –NH3+, the carboxylic group will remain as –COOH (aa will migrate towards the cathode). As pH is increased, –COOH (from some fraction of aa) ionises. When the pH is equal to the pKa1 the amino acid exists as a 50:50 mixture of the cationic and zwitter ionic forms. As pH is further increased more cationic form converts to the zwitterionic Can donate & accept H+ i.e amphoteric nature therefore aa are ampholytes

  23. - Adding more base results in continued ionization of the carboxylic acid group until the zwitter ionic form is the predominant form of the amino acid in solution. By the addition of more base, the pKa of the amino group is reached and at this point the amino acid exists as a 50:50 mixture of the zwitter ionic form and the anionic form. As the pH is increased further the amino group continues loses its proton and ultimately, at high pH (pH ~ 12.0), the anionic form is the predominant form in solution. At pH>~9.6 the amino group will be as –NH2, the carboxylic group will remain as -COOˉ (aa will migrate towards the anode). • So at physiological pH 6.8 - 7.4, the –COOH group exist as COO¯, and the –NH2as–NH3+. Therefore all aa are double-charged structure or zwitterion in this pH region. The pH at which they exist as “whole” zwitterion i.e the molecule carries no electrical charge, or the negative and positive charges are equalis called Isoelectric point (Ip) or Isoelectric pH .

  24. aa Actual structure CH3-CHCOOH CH3CH COO¯ NH2 N+H3 Neutral un-charged NOT THIS Zwitterion. Neutral but charge - Eachaahas its Ip value. At Ip: i) aa is double-charge (zewtterionic) i.e +ve & -ve, amount of positive charge exactly balances the amount of negative charge so net charge is 0 (electrically neutral). ii) it does not move/migrate in electric current iii) the molecule has minimum solubility. iv) Ip of all aa lie in the range of pH 6.8 - 7.4 Isoelectric pH of an aa solution is given by: pH = ½ (pK1 + pK2) High pH region Low pH region

  25. For aliphatic aa 50% as cationic 50% aszwitterion 50% as anionic 50% aszwitterion E.g The pH profile of an acidic solution of alanine when the solution is titrated with a strong base, NaOH.

  26. Physical properties: - colourless crystalline; soluble in water/polar solvents. Tyrosine is soluble in hot H2O - have high m.pt >200oC - have high dielectric constant and high dipole moment - molecules have minimum solubility in water or salt solutions at the Ip pH and often precipitate out of solution.Why? At Ip aa is in zwitterionic form therefore non-polar. Hence no interaction with polar water molecules • Chemical properties: involve –COOH & involve –NH2 i) involve –COOH • decarboxylation or formation of amine & CO2 eg. histadine histamine + CO2 tyrosine tyromine + CO2 tryptophan tryptamine + CO2 lysine cadaverine CO2 Glutamic gamma amino butyric acid (GABA) + CO2

  27. • Amide formation : α-COOH of 1 aa reacts with α-NH2 of aa behind to form a peptide bond or CO—NH bridge eg in peptides and proteins Amide formation (at 2nd —COOH) aspartic + NH3 asparagine glutamic + NH3 glutamine (than N donated for N.A synthesis) ii) involve –NH2: ● formation of carbamino compound –NH2 + CO2 –NH-CO2H eg transport of CO2 by hemoglobin from tissue to lung Hb–NH2 Hb–NH-CO2H (carbamino-Hb) ● Transamination eg in metabolism pathway RCHCOOH + R’CCOOH RCCOOH + R’CCOOH NH2 O O NH2 ● oxidative Deamination eg. in metabolism pathway RCHCOOH RCCOOH + NH3 NH2 O ‼ ‼ ‼

  28. Contributing properties from R groups • When R group is plain hydrocarbon (gly, ala, leu, isoleo, val) the a.a interact poorly with water. • * When R group have functional groups capable of hydrogen bonding e.g -OH ( Ser, thr, tyr) ; -COOH (asp and glu), these a.a are Hydrophilic or ‘water-loving’ so easily interact with water. • Ester Formation by –OH of serine -OH + H3PO4 phosphoproteins -OH + polysaccharide O-glycoprotein • * When R group have functional group –COOH ( asp , glu) the a.a can exist as –ve molecule physiological pH and can form ionic bonds with basic amino acids. • When R group have functional group –NH2/ -NH(lys and hist) , these a.a are +ve charged at physiological pH and can form ionic bonds with acidic amino acids. • The sulfhydryl group of cysteine is highly reactive. -Oxidation of two molecules of cysteine forms cystine. The 2 molecules is linked by a disulfide bond/bridge. The reaction is reversible oxidation

  29. Transmethylation methyl group of methionine may be transferred to an acceptor to become intermediates in metabolic pathway • Formation of S-S bridge. sulfhydryl (-SH) group of cysteine can form the S-S bond with another cysteine residue intrachain or interchain 2 cysteines cystine Function of R groups is also very significant in function of peptides and Proteins. Few examples: a) The hydrophobic aa will generally be found in the interior of proteins shielded from direct contact with water b) The hydrophilic aa will generally be found in the exterior & active centre of enzyme. c) The imidazole ring of histidine acts as proton donor or acceptor at physiological pH hence it is normally found in active site of enzyme, in hemoglobin (RBC).

  30. Few aa are origin/starting molecules for important compounds or amino acid derived molecules: • Glutamic acid Gammaaminobutyric acid (GABA) • Tyrosine dopamine. these are neurotransmitters. • Histidine histamine, a mediator of allergic reactions • Tyrosine thyroxine, a thyroid hormone • Serine cycloserine an anti-tuberculous; azaserine, an anti-cancer molecule • Arginine ornithine and citrulline, intermediates in urea cycle

  31. 2. Structure and function of proteins To enable to: • Describe the formation of peptide bonds • Describe the four levels of protein organization with reference to primary, secondary, tertiary and quaternary structure of proteins using haemoglobin as example • Explain how structure of protein determines its function by looking at examples • Differentiate between globular and structural proteins with examples eg immunoglobulin, hemoglobin, collagen, keratin etc • Describe the functions of protein • Relationship between structural protein and its function in health and disease.

  32. Proteins: Biological Functions • as biological catalysts of the chemical reactions that occur within the cell examples: i- starch maltose + shorter chain starch ii- protein amino acids + peptide chain iii- triglyceride f f a + mono + di iv- ATP ADP +Pi α-amylase trypsin lipase glycerides phosphatase

  33. As regulatory proteins.These proteins regulate the activities of the cell and the ability of other proteins to carry out their cellular functionin regulating overall metabolism, growth, development, and maintenance of the organism eg peptide and protein hormones; allosteric enzyme; gene inducers & repressors. • As transporter molecules eg. hemoglobin; GLUT,SGLUT i- hemoglobin transport O2 from tissue to lungs; myoglobin transport O2 intracellular ii- GLUT transport glucose/galactose from intestinal to blood, iii- SGLUT transport glucose from intestinal to blood. • As storage proteinseg myoglobin, stores O2 in muscle tissue

  34. A peptide bond (amide bond): - feature bonds between amino acids (aa) in polypeptides and proteins. - is formed when the carboxyl group of one aa molecule reacts with the amine group of the other aa molecule in front of it, thereby releasing a molecule of water (H2O). - this is a dehydration synthesis reaction or condensation reaction, - the resulting CO-NH bond is called a peptide bond, and the resulting molecule is an amide. The four-atom functional group -C(=O)NH- is called an amide group or (in the context of proteins) a peptide group. - living organisms employ enzymes to form peptide bonds. eg.during translation process. - When two amino acids are linked together, the product is called a dipeptide andwhen the product is of threeamino acids then it is tripeptide

  35. Peptide bond ―C―N O H • feature bonds between amino acids (aa) in polypeptides and proteins. • is a bond formed when a carboxylic group reacts with an amino group instantaneously eliminating a molecule of H2O • this is a dehydration synthesis reaction or condensation reaction, • the resulting CO-NH bond is called a peptide bond, and the resulting molecule is an amide. • The four-atom functional group -C(=O)NH- is called an amide group or (in the context of proteins) a peptide group. • living organisms employ enzymes to form peptide bonds. eg.during translation process. • When two amino acids are linked together, the product is called a dipeptide andwhen the product is of threeamino acids then it is tripeptide

  36. Structure organization in proteins • Primary Structure (or primary level of organization) Definition. Is "The sequence of amino acids in the polypeptide chain.", The N-terminal on the left and C terminal on the right. • chain has 50 to 2000 amino acid residuesso it is a polypeptide • The residues are joined by peptide bonds • Changes in the primary structure can alter the proper functioning of the protein.eg offcoded of 2 amino acid in the protein of the glycoprotein in RBC results in MN blood group

  37. In actual chain these R groups will be the various side chains Peptide bond C-terminal N-terminal

  38. Effect of surrounding pH on the structure At neutral pH Protein with basic aa will have overall positive charge. And thatwith acidic aa will have overall negative charge

  39. cont Effect of surrounding pH on the structure

  40. Secondary structure: There are two types : the α-helix and the β-pleated sheet. • The attraction between the R groups can occur within the same chain (case I) or between chains lying next to one another (case II). Case I leads to formation of weak bonds eg hydrogen bonds ; R-R attraction etc. The hydrogen bonds is "Intrachain Hydrogen Bonding" which is between the hydrogen and oxygen atoms of the amino acid backbone. These intrachain weak bondings can cause the chain to twist into a "right handed" coil or α-helix. Case II leads to formation of β-pleated sheet. Such “secondary structure α-helix ” often predominate in "globular proteins“ and β-pleated sheet predominate in fibrous proteins.

  41. Globular proteins are (i) compactly folded and coiled somewhat spherical. The molecule’s apolar a.a bound towards the molecule interior and the polar a.a bound towards the molecule exterior allowing dipole-dipole interaction with the solvent. (ii) Soluble in aqueous medium giving colloidal solution (iii) Play numerous functions, as: i) enzymes eg esterases ii) messengers/hormones eg. Insulin iii) transporter of molecules across membran iv) storage eg myoglobin ** α-helix: "alpha" means, looking down the length of the spring, the coiling is happening in a clockwise direction β- pleated sheets: the chains are folded so they lie alongside each other H2 bond β-pleated , anti-parallel (arrows running in opposite direction

  42. Myoglobin - first globular protein whose structure was analysed by X-ray diffraction by protein crystals. The periodic repeats characteristic of alpha helix were recognised, and the structure shown to have 70% of the polypeptide is alpha-helical. - it is O2 storage site in muscle tissue. - It is also intracellulartransporter of O2. - Its tertiary (3-D) structure consists of a 8 α-helices which fold to make a compact globular protein. - the side facing the interior having amino acids with hydrophobic side-chains ie. hydrophobic groups are on the inside of the protein. The side facing to outside having polar side-chains ie. hydrophillic groups are on the outside of the protein,facing the aqueous environment.

  43. Myoglobin Structure Reference: J.Mol. Biol.142, 531-554. A representation of the 3D structure of the myoglobin protein. Alpha helices are shown in colour, and random coil in white, Heme with Fe2+/3+

  44. β-pleated sheet - the β-pleated sheet forms when the hydrogen atoms of the amino group and the oxygen atoms of the carboxyl group of amino acids on two chains (or more) lying side-by-side forms hydrogen bond. - Closely associate to structural/fibrous proteins - the protein chains are in associate to form long fibers - elongated or needle shaped - possess minimum solubility • resist digestion - The β-pleated sheet structure is often found in many structural proteins, eg "Fibroin", the protein in spider webs; Keratin- a structural protein found in hair and nails, skin, and tortoise shells

  45. Fibrous proteins are more filamentous or elongated, play only structural funtions. Also known as scleroproteins. Found only in animals. Are water-insoluble. Used to construct connective tissues, tendons, bone matrix, muscle fibers. Examples are keratin (hair; tough and hard bud not mineralized structure as in reptiles) , collagen ( long chains, tied into bundles, has great tensile strength). Its degradation leads to wrinkles that accompanying aging.

  46. "Tertiary" Structure: a 3 dimensional chain arrangement, • the way the whole chain (including the secondary structures) folds itself into its final 3-dimensional shape • is held together by interactions between the side chains - the "R" groups. Interactions such as: ionic; van der Waals (hydrophobic-hydrophobic); H-bonds; S-S bridge OR • When "proline", an oddly shaped amino acid occurs in the polypeptide chain a "kink" in the a-helix develops. Kinks can also be caused by repulsive forces between adjacent charged R groups. These kinks create a 3 dimensional chain arrangement This 3 dimensional shape is also held together by weak hydrogen bonds "disulfide" bonds between two amino acids of cystine ("covalent") disulfide "bridges" (linkages) cystine -- s -- s – cystine. These strong covalent bonds hold the protein in its specific 3D shape. The 3D shape creates "pockets" or "holes' in the surface of the protein which are very important in enzyme function

  47. Cystinyl α-helix random coils pleated sheets

  48. Quaternary Structure of Proteins • 2 or more 3 dimensional tertiary proteins and sticking them together to form a larger protein. Many enzymes and transport proteins are made of two or more parts. • only exists, if there is more than one polypeptide chain present in a complex protein • Hemoglobin: an oxygen carrying protein in red blood cells which is made of 4 parts.