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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
- digestion and absorption - metabolism - metabolic disorder disease Amino Acid : - absorption and metabolism

4 Expression of Concentration
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 Volume of solution (dm3 or liter)
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 M = moles of solute (mol) Volume of solution (dm3 or liter) moldm-3 molL-1 m = moles of solute (mol) mass of solvent (kg)

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 be confused 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 μg cholecalciferol/ergocalciferol Vitamin E: 1 IU is the biological equivalent of about mg d-alpha-tocopherol (2/3 mg exactly), or of 1 mg of dl-alpha-tocopherol acetate

9 III. a) Making Dilutions process 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 solute M1 V1 = M2 V2

10 d) supersaturated solution
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 hypertonic or hypotonic Hypertonic solution contain a high concentration of solute relative to another solution on 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 CH3CO CH3 CZRCO2-

13 Ka = [H3O+] [CH3COO-] Derivation of Henderson–Hasselbalch equation
[CH3COOH] [H3O+] = Ka [CH3COOH] [CH3COO-] X each by (- log) …… – log [H3O+] = – log Ka – log [CH3COOH] pH = pKa – log [CH3COOH] pH = pKa + log [CH3COO-] 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 = = 0.005 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
Amino acid (a.a) 20 altogether = std aa all aa share a general formula R-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 COOH

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 Nonessential Isoleucine Alanine Leucine Asparagine Lysine Aspartate Methionine Cysteine* Phenylalanine Glutamate Threonine Glutamine* Tryptophan Glycine* Valine Proline* Serine* Tyrosine* Arginine* Histidine* * Essential in certain cases. 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+H ―NH 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 , the –COOH group exist as COO¯, and the –NH2 as –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 equal is called Isoelectric point (Ip) or Isoelectric pH .

24 - Each aa has its Ip value. At Ip:
aa Actual structure - Each aa has 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 Isoelectric pH of an aa solution is given by: pH = ½ (pK1 + pK2) CH3-CHCOOH CH3CH COO¯ NH2 N+H3 Neutral un-charged NOT THIS Zwitterion. Neutral but charge High pH region Low pH region

25 For aliphatic aa 50% as cationic 50% as zwitterion 50% as anionic 50% as zwitterion 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 + NH asparagine glutamic + NH glutamine (than N donated for N.A synthesis) ii) involve –NH2: ● formation of carbamino compound –NH2 + CO –NH-CO2H eg transport of CO2 by hemoglobin from tissue to lung Hb–NH Hb–NH-CO2H (carbamino-Hb) ● Transamination eg in metabolism pathway RCHCOOH + R’CCOOH RCCOOH + R’CCOOH NH O O NH2 ● oxidative Deamination eg. in metabolism pathway RCHCOOH RCCOOH NH3 NH 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 + H3PO 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 i- starch maltose + shorter chain starch
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
As regulatory proteins. These proteins regulate the activities of the cell and the ability of other proteins to carry out their cellular function in 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 proteins eg 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 and when the product is of three amino acids then it is tripeptide

35 feature bonds between amino acids (aa) in polypeptides and proteins.
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 and when the product is of three amino acids then it is tripeptide

36

37 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 residues so 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

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

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

40 Effect of surrounding pH on the structure
cont Effect of surrounding pH on the structure

41 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.

42 (ii) Soluble in aqueous medium giving colloidal solution
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

43 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 intracellular transporter 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.

44 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+

45 β-pleated sheet - Closely associate to structural/fibrous proteins
- 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

46 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.

47 "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

48

49 Cystinyl α-helix random coils pleated sheets

50 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.

51 Structural Level of Proteins

52 Denaturation or Loss of 3-D shape
denaturing agents: Temperature> 40oC; mineral acids; salts. eg. when heated, protein can unfold or "Denature". This loss of three dimensional shape will usually be accompanied by a loss of the proteins function. If the denatured protein is allowed to cool it will usually refold back into it’s original conformation.

53 Protein metabolism denotes the various processes responsible for the
(i) biosynthesis of proteins from amino acids. (ii) catabolism the breakdown of proteins by /proteolysis liberating of amino acids. That is, comprises of I- Protein metabolism (synthesis and breakdown) II-Amino Acid metabolism (synthesis and breakdown) WILL PROCEED WITH Protein metabolism (synthesis and breakdown)

54 PROTEIN SYNTHESIS proteins of one organ are similar but differ from that of another organ. That is, each chain is characterized by a specific sequence of a.a. How is this special feature achieved? The sequence of a.a in a particular chain is ensured through the following units and process: translation; Codons; transciption tRNA; mRNA;

55 Translation is “process of protein synthesis”
Translation is “process of protein synthesis”. It is translating genetic messages into the primary sequence of a polypeptide. tRNA carries a specific amino acid to the matching position along the mRNA template. It can be divided into 4 stages: Activation, Initiation, Elongation and Termination, each regulated by a large number of proteins and coactivators. It occurs in cytoplasm. Codon: a sequence of 3 nucleotide in DNA that codes a single a.a transcription : synthesis of a single strand messenger RNA (mRNA) by transcribing the sequence of the nucleotide in the template DNA/genom. The reaction is catalyzed by RNA polymerase . The template DNA is “unzipped” by enzyme helicase prior to the transcription.

56 tRNA is transfer RNA that carries an a
tRNA is transfer RNA that carries an a.a to the mRNA to be incorporated into the peptide chain. mRNA is a type of RNA that encoding the sequence of the protein in the form of a trinucleotide code . The specific sequence of the nucleotide is accomplished through transcription.

57 The synthesis process/translation
Activation: the correct amino acid (AA) is joined to the correct tRNA. The AA is joined by its carboxyl group to the 3' OH of the tRNA by an ester bond. The anti-codon determines the correct AA. Initiation: involves the small subunit of the ribosome binding to 5' end of mRNA with the help of initiation factor (IF), Elongation occurs when the next aminoacyl-tRNA (charged tRNA) in line binds to the ribosome along with GTP and an elongation factor. Termination of the polypeptide happens when the A site of the ribosome faces a stop codon (UAA, UAG, or UGA).This activates release factor which then causes the release of the polypeptide chain.

58 TRANSLATION in diagrame :
LOADED tRNA COMPONENTS PRESENT IN THE PROCESS Aminoacid carried anticodon codon RIBOSOME mRNA

59 TRANSLATION 1º AMINOACID: Methionine (AUG) in site P.
The newly made mRNA (transcription) leaves the nuceus and binds with the ribosome in the cytoplasm. ONE codon is exposed at site P and another codon at site A A tRNA with a complementary codon in its anticodon site will bind with the codon at site P, bringing an aminoacid. 1º AMINOACID: Methionine (AUG) in site P.

60 TRANSLATION Even though every protein begins with the Methionine amino acid, not all proteins will ultimately have methionine at one end. If the "start" methionine is not needed, it is removed before the new protein goes to work (either inside the cell or outside the cell, depending on the type of protein synthesized)

61 TRANSLATION PEPTIDIC BOND IS FORMED 2º AMINOACID:
Glycine (only in this case) in site A. A PEPTIDIC BOND IS FORMED

62 TRANSLATION Growing polypeptide
NO aminoacid is added. Its the END of the polypeptide! STOP codon

63 PROTEIN CATABOLISM Has various indication:
Comprises of Digestion and Absorption Is carried out via proteolysis is the directed degradation (digestion) of proteins by cellular enzymes called proteases (various kinds) releasing peptide/A.A The digestion of proteins from foods as a source of amino acids (aas) The aas constituting “aa pool” are metabolized further ( aa catabolism)

64

65 DIGESTION & ABSORPTION
Source of proteins that come in the diet: - animal eg milk, dairy products, meat, fish, eggs, liver - vegetable sources eg cereals, pulses, peas, nuts and beans In mouth: no proteolytic enzyme so the proteins are unchanged but the size(food) becomes smaller due to mastication and chewing. Food bolus travels down and reaches stomach and meet gastric juice In stomach ( pH 1-2 maintains by HCl) : attack by pepsin, renin, gelatinase and gastricin ( enzymes in the gastric juice). All these enzymes attack internal peptide bonds. - Pepsin( a endoproteinase) acts on : Proteins proteoses + peptones Casein(milk) paracasein + proteos (whey proteins) paracasein + Ca calcium paracasein (insoluble) - gastricin ( a proteinase) - gelatinase: gelatine polypeptide

66 In small intestine : duodenum, jejunum, ileum
Food bolus meet pancreatic juices. Enzymes in pancreatic juices : Trypsin ( a proteolytic enzyme) Chymortypsin Carboxy peptidases ( 2 types: A and B) are exopeptidases; splits one amino acid at a time fr free end. Elastases : a serine protease Collagenases act on protein present in collagen/connective tissue yielding peptide - Jejunum-ileum Food remnant meet intestinal juice. Enzymes in intestinal juices: Amino peptidase: peptides tripeptides Enteropeptidase/Enterokinase Prolidase: acts at terminal proline Di and tri-peptidase: Di and tri-peptide  amino acids

67 Absorption Of Amino Acids
Absorption is by active transport Site of absorption is - ileum and distal jejunum: amino acids - duodenum and proximal jejunum: di and tri-peptides After absorption, amino acids and di and tri-peptides (if any) are carried by portal blood to liver, partly : i- are taken up by liver cells ii- enter the systemic circulation (made up part of aa pool), diffusing throughout body fluid & taken up by tissue cells. ( The body's circulatory system has three distinct parts: pulmonary (the lungs) circulation, coronary (the heart) circulation, and systemic (the rest of the system) circulation. Each part works independently in order for them to all work together)

68 The aa will be used to synthesize: tissue proteins; enzyme; hormones
3 states relates to aa pool -cell : i- dynamic equilibrium amnt of aa taken-up = amnt of aa loss ii- cell waste  amnt of aa taken-up < amnt of aa loss iii- cell grows  amnt of aa taken-up > amnt of aa loss Regulatory of Amino Acid If amino acids are in excess of the body's biological requirements, they are metabolized to glycogen or fat and subsequently used for energy metabolism. If amino acids are to be used for energy their carbon skeletons are converted to acetyl CoA, which enters the Krebs cycle for oxidation, producing ATP

69 Summary: Digestion & Absorption
Aas available for use in metabolic processes come from dietary protein and breakdown of tissue protein by proteolysis. Digestion (dietary protein) occurs in stomach as well as intestine. In stomach, digested by pepsin, in intestine and duodenum by a group of enzymes, protease (trypsin. Chymotrypsin and carboxypeptidase) These liberated aas are absorbed into cells and are collectively referred as “aa pool” ## Amino acids are transported into cell by various transport mechanisms involving membrane-bound transport proteins. Ingested protein→ digested → aa → absorbed (aa pool)

70 Assimilation of Amino Acids
Tissue protein Dietary protein enzymic proteolysis e.g trypsin/pepsin OUTSIDE CELL INSIDE CELL A A pool ─ C skeleton synthesis ─NH2 protein Precursors for other molecules Excreted as urea & uric acid energy New AA N containing molecules Healthy & in young subject >> aa breakdown

71 Amino acid synthesis is the set of metabolic pathways /processes by which the various amino acids are produced from direct incorporation/ combination (I) of –NH2 group OR (II) of ammonium ion NH4+ with other compounds found in the organism’s diet or growth media I –NH2 group is incorporated into α-keto acid through 2 types of reactions: i- non-reductive transamination i) glutamate/aspartate as –NH2 donor ii) glutamine/asparagine as –NH2 donor iii) branch chain aa as –NH2 donor ii- reductive transamination II- ammonium ion NH4+ is incorporated α-keto acid. through - Reductive amination - non-reductive amination

72 Non-reductive transamination characteristics:-
Reaction of Glutamate (or Aspartate) and an α-keto acid or BCAA. -NH2 is transferred from Glutamate/ Aspartate to an α-keto acid. ( Glutamate/ Aspartate/asparagine is -NH2 group donor; α-keto acid supplies C-skeleton) ** glutamate as -NH2 group donor is more regular Reaction is catalysed by i) enzyme aminotransferase or transaminase ; ii) required co-enzyme pyridoxal-5’-phosphate (PLP) O R1—C—C--O‾ R1—CH—C--O‾ α-ketoglutarate O O NH3 Acceptor α-ketoacid New AA referred as pair

73 Non-reductive transamination: Examples
i) Glutamate and an α-keto acid (pyruvate) NH CH CH3 OOC-CH2-CH2- CH C=O HC─NH OOC-CH2-CH2C=O COOH COOH COOH COOH Glutamate pyruvate alanine α-ketoglutarate ii) Aspartate and an α-keto acid (pyruvate) pair NH CH CH3 OOC-CH2-- CH C=O HC─NH OOC--CH2C=O COOH COOH COOH COOH aspartate pyruvate alanine oxaloacetate

74 iii- asparagine and an α-keto acid (pyruvate)
COOˉ CH3 COOˉ CH3 + CH-NH2 COOˉ CO-CH2-CH C=O NH NH COO COO-CH2-CH NH3+ Pyruvate Aspatate Asparagine Alanine Transaminase ** Aspartate transaminase or aspartate aminotransferase is an enzyme associated with liver parenchymal cells.

75 enzyme: glutamine synthase (GS)
Non-reductive transamination (in skeletal muscle). enzyme: glutamine synthase (GS) Glutamate + BCAA → glutamine + α-keto acid ( BCAAs are comprised of valine, leucine, and isoleucine) COO COO COO COO OOC-CH2-CH2-CH + (CH3)2CH-CH O=C-CH2-CH2-CH + (CH3)2-CH-C NH NH NH NH O Glutamate valine glutamine BC α-oxoacid

76 Enzyme Transaminase/Aminotransferase
requires co-enzyme pyridoxal-5’-PO4 , abbreviated (PLP). a derivative of vitamin B6 R of Lysine

77 PLP attaches to the active site of enzyme by noncovalent interaction and a Schiff base aldimine ( condensation of ε-amino of lysine residue and aldehyde group of PLP) is formed. amino acid substrate becomes bound to PLP via the α-amino group in an imine exchange reaction. bond 1 breaks leaving –NH2 on the co-enzyme to be transferred to an α-keto acid, [ Vitamin B6 is involved in the metabolism (especially catabolism) of amino acids, as a cofactor in transamination reactions. This is the last step in the synthesis of nonessential amino acids and the first step in amino acid catabolism. Vitamin B6 is a mixture of pyridoxin derivatives. PLP is 1 of them].

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79 Glutamate in transamination: (pyruvate/alanine pair)
(oxaloacetate/aspartate) NH CH CH3 OOC-CH2-CH2- CH C=O HC─NH OOC-CH2-CH2C=O COOH COOH COOH COOH Glutamate pyruvate alanine α-ketoglutarate NH CH2 COO CH2COO OOC-CH2-CH2- CH C=O HC─NH OOC-CH2-CH2C=O COOH COOH COOH COOH Glutamate oxaloacetate aspartate α-ketoglutarate GS **(in skeletal muscle) Glutamate + BCAA → glutamine + α-keto acid BCAAs are comprised of valine, leucine, and isoleucine

80 Reductive Transamination
Glutamine, asparagine transfer the amide nitrogen to oxo (or keto) acid to form a new amino acid. 2-oxoglutarate is –NH2 receptor and glutamine is –NH2 donor The enzyme GOGAT is NADPH dependent glutamine + 2-oxoglutarate + NADPH + H+ ---> 2 glutamate + NADP+ GOGAT GOGAT: enzyme glutamine oxoglutarate amidotransferase

81 II- Incoporation of NH4+ ion:
i) Reductive amination reaction of α-ketoglutarate with NH4+ leading to formation of glutamate (in mitochondria & cytoplasm). α-ketoglutarate is –NH2 acceptor catalysed by glutamate dehydrogenase, the enzyme is NADH dependent reaction is reversible  i.e the reverse pathway is a primary means of producing NH4+ for N excretion. The enzyme is driven toward right when excess NH4+ is present NH4+ is from oxidative deamination of glutamate (in extrahepatic tissue)

82 Reductive Amination : left - right
GD + NH4+ + NADH + H+ + NAD+ + H2O Enzyme: Glutamate Dehydrogenase NH3 + H+ Reductive Amination : left - right (Oxidative Deamination : right  left)

83 ii) Non-reductive amination or amidation
Glutamate or aspartate react with NH4+ to form glutamine, (asparagine) catalyze by glutamine/ asparagine synthetase respectively. Sites : liver, brain , kidney, muscles & intestine This rxn forms the path by which cell rid off excess NH4+. ** NH4+ at high conc may be toxic to certain cell e.g brain cell. Glutamine is non toxic.

84 COO- COO- Non-reductive amination or amidation From excess aa pool
CO-CH2-CH2- CH COO-CH2-CH2- CH + ATP + NH4+ + ADP NH2 NH3+ + Pi NH3+ Glutamine Glutamate Glutamine Synthetase (GS) COO- COO- CO-CH2-CH NH NH3+ COO-CH2- CH + ATP + NH4+ + ADP + Pi NH3+ Asparagine Synthetase Asparagine Aspartate

85 Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer`s disease.

86 Oxidative deamination
glutamate α-keto acid GD transamination Reductive amination New aa NADH NH4+ α-ketoglutarate Oxidative deamination glutamate

87 C skeleton of all non-essential aa are derivatives of:
Glycerate -3-phosphate Pyruvate Α-ketogluterate Oxaloacetate But Tyrosine from essential aa phenylalanine On basis of common precursor Ξ similarities in their synthetic Pathway, aa can be grouped into 5 families.

88 glutamate family= synthesis of glutamate, glutamine, arg, pro.
- C skeleton derive fr α-ketoglutarate serine family = synthesis of serine, glycine, cystein - C skeleton derive fr glycerate-3-phosphate aspartate family = synthesis of aspartate, lysine, methionine, asparagine, threonine - C skeleton derive fr oxaloacetate pyruvate family = synthesis of alanine, valine, leucine, isoleucine - C skeleton derive fr pyruvate aromatic family = synthesis of *phenylalanine, tyrosine, *tryptophan *EAA

89 Glutamate family - key substrate is α-ketoglutarate fr TCA Glutamate is produced by GD and is the principle rxn of fixation of NH3 in bactria glutamine is produced by ATP-requiring +n of NH3 to glu and the rxn fnc as a major means of assimilating of NH3 fr environment Regulation of this family is controlled by repression of mRNA and feedback inhibition: by prolin and arg

90 Regulatory of Amino Acid
If amino acids are in excess of the body's biological requirements, they are metabolized to glycogen or fat. If amino acids are to be used for energy their carbon skeletons are converted to acetyl CoA, or other metabolites intermediates (pyruvate, oxaloacetate, Succinyl-coA ) which then enters the Krebs cycle for oxidation, producing ATP.

91 Catabolism of AA Generally involves : Removal of amino group
Disposal of amino group to final compounds urea([NH2]2CO) and ammonia (NH3); also incoporated into other molecules Utilization of C skeleton by channeling into TCA through which they are converted to final products carbon dioxide (CO2), water (H2O), ATP, or degraded into a variety of metabolite intermediates which then enter synthesis pathway of other compounds Decarboxylation - one carbon metabolism

92 glutamate + NAD+ −− NH+4 + α-ketoglutarate
Removal of amino group Occurs by - transamination - oxidative deamination (only happens with glutamate ) catalyses by glutamate dehydrogenase glutamate + NAD+ −− NH α-ketoglutarate Transamination ( largely occurs in cytosol of liver cells) is the transfer of the nitrogen (the amino) group of an L-a.a to α-ketoglutarate forming L-glutamate. The reaction is catalysed by transaminase and it requires co-enzyme pyridoxal-5’-PO4(see earlier section for detail mechanism). Glutamate may undergo another transamination, transfering –NH2 to another α-ketoacid i.e glutamate becomes -NH2 carrier

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94 Oxidative Deamination

95 Oxidative Deamination (O.xdn)
reaction is prevalent when proteinintake> proteinsynthesis => aa from“aa pool” undergoes degradation. The N- in aa is removed by deamination rxn and converted to ammonia which is toxic, therefore need to be detoxified and excreted. Is :L-glutamate + NAD+ −− NH+4 + α-ketoglutarate happens only with glutamate catalyses by glutamate dehydrogenase GD. It occurs in liver & in most extrahepatic tissue.

96 * N of amino group made available for excretion by rxn .
In muscle cell ( no GD) any excess aa transfer its -NH2 to α-ketoglutarate to form L-glutamate (transamination). L-glutamate undergoes transamination with pyruvate catalyse by alanine transaminase to give alanine + α-ketoglutarate. Alanine carries by blood to liver, (alanine cycle) . In liver, alanine + α-ketoglutarate react catalysed by alanine transaminase reforming L-glutamate + pyruvate as alanine transaminase rxn is reversible. Then L-glutamate undergoes Oxidative deamination. Pyruvate can be diverted to gluconeogenesis. This process is refered to as the glucose-alanine cycle and NH+4 moves onto urea cycle which is also known as ornithine cycle, be converted to urea. Urea is transferred through the blood to the kidneys and excreted in the form of urine.

97 -NH2 in Muscle NH4+ (liver)
Alanine Cycle -NH2 in Muscle NH4+ (liver) Transported to liver for Oxidative deamination glutamate New α-keto acid alanine Alanine transamination transamination excess aa pyruvate α-ketoglutarate alanine glutamate GD NH4+ + NADH H2O + NAD+ Liver Alanine transamination To urea cycle α-ketoglutarate pyruvate α-ketoglutarate

98 Deamination is also an oxidative reaction
•occurs under aerobic conditions in all tissues but especially the liver. During oxidative deamination, an amino acid is converted into the corresponding keto acid by the removal of the amine functional group as ammonia and the amine functional group is replaced by the ketone group. The reaction is catalysed by glutamate dehydrogenase which is allosterically controlled by ATP and ADP. ATP acts as an inhibitor whereas ADP is an activator. The ammonia eventually goes into the urea cycle. Oxidative deamination occurs primarily on glutamic acid because glutamic acid was the end product of many transamination reactions.

99 GD The glutamate dehydrogenase (GD) is allosterically controlled by ATP and ADP. ATP acts as an inhibitor whereas ADP is an activator.

100 Summary of Urea Cycle Occurs in liver cells
Is a 5 steps cycle: 1 step in mitochondria 4 steps in cytosol Main substrates: NH3, CO2 and Aspartate. In the matrix of mitochondria occurs CPS I and OTC catalysed rxn, CPS rxn uses 2ATP and reaction is irreversible Citrulline Ornithine occur in cytosol, in 4 steps -Citrulline is tranported across the inner membrane by a carrier neutral aa. - enzymes are arginosuccinate synthase, arginosuccinate lyase and arginase Urea transferred to kidney through blood and excreted as urine

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105 Fate of A.A Nitrogen Excreted in the form of urea (urine)
Transferred to specific α-keto acids (of the TCA intermediates) to form new a.a. This can be represented in the form of α-keto acids / aa pair eg: α-ketoglutarate/glutamate; pyruvate/alanine; aspartate/oxaloacetate pair. Incorporated into skeleton of non amino acid molecules => aa derived compound

106 Derived AA Compounds What are derived amino acid compounds?
They are compounds that contain N- atom, S- atom or part of aa structure as part of their molecular structure .Can be divided into 2 groups: alkaloids (in plants) & animal related. Animal related and specific parent aa eg. Glutathione (GSH), Serotonin and Histamine, Heme, GABA , DNA bases Why the synthesis occurs? These molecules are synthesized because they are important to the body. The synthesis process

107 Parent aa derived compd glutamate Glutathione(GSH) tyrosine Dopamine melanine GABA tyroxine Epinephrine/ norepinephrine Serine ethanolamine Choline Leucine β-OH-β-methylglutaryl-CoA Betaine Lysine carnithine tryptophan Serotonine, melatonine Histidine Histamine, Carnosine, anserine

108 serotonin Fncn to influence the functioning of the cardiovascular, renal, immune, and gastrointestinal systems Any disruption in the synthesis, metabolism or uptake of this neurotransmitter has been found to be partly responsible for certain manifestations of schizophrenia, depression, compulsive disorders and learning problems. Synthesis:

109 Function of some AA derived compounds
As neurotransmitter : GABA, dopamine, serotonin, Sleep inducing : melatonin Carrier : carnithine As hormone: tyroxine, Dilating/constriction of blood vessel: histamine Exhibit multifunctions: GSH - acts as reducing agent in NA and eicosanoids synthesis - maintain the sulfahydryl grp of enzymes & other molecules in reduced state - promotes aa transport - protect cells fr radiation, O2 toxicity and environmental toxins

110 Utilization of the C-skeleton
The C-skeleton of the standard amino acids are degraded to seven common metabolic intermediates such as Acetyl-coA; Acetoacetyl-CoA; pyruvate; Oxaloacetate, α-ketoglutarate, Succinyl-CoA and fumerate. Those aa are referred to different names depending to the class to which the final product are classified: i) degraded to acetyl-CoA and AceAcetyl-CoA are referred to as KETOGENIC because the intermediates lead to either fatty acids or ketone bodies.eg Lys and Leu

111 ii) degraded to pyruvate; α-ketoglutarate, Succinyl-CoA, Oxaloacetate, and fumerate are referred to as GLUCOGENIC because they are intermediates of gluconeogenesis. All except Lys and Leu are pure or partly glucogenic Those that yield acetyl-CoA are divided into 2 groups. a) Those that yield pyruvate as intermediate: Ala, Cys, Gly, Ser and Thr b) Those that do not yield pyruvate as intermediate: Phe, Lys, Leu Trp and Tyr

112 utilization of the C-skeleton

113 Decarboxylation of amino acid
is effected by decarboxylase enzyme, PLP dependent Products are alkylamine + CO2 . The alkylamine are neurotransmitters There are 4 aa decarboxylase enzymes: Aromatic L-amono acid decarboxylase (is a group of enzymes); L-glutamate decarboxylase (GAD); lysine decarboxylase (LDC); histidine decarboxylase (HDC)

114 GABA is a neurotransmitter in brain GAD (GABA)
HDC HOOC-CH2-CH2-CH(NH2)-COOH ───→ CO2 + HOOC-CH2-CH2-CH2NH2 GABA is a neurotransmitter in brain GAD (GABA) Aromatic L-aa decarboxylase synonyms to DOPA decarboxylase, tryptophan decarboxylase, 5-hydroxytryptophan decarboxylase, AAAD. tryptophan ───→ tryptamine + CO2 Tryp D

115 A.As Metabolic Disorder Diseases
Are diseases resulted from disorders of a.as processing/metabolism due to Inherited/genetic defects that cause deficiency of certain enzymes for i) the breakdown of amino acids or ii) the body's ability to get the amino acids into cells or iii) Amino acid Transport Symptoms of disease appear early in life - Generally are autosomal recessive that is why only small number of man suffers.

116 Inherited metabolic disorder ( I.M.D) :
Oculocutaneous albinism Tyrosinemia of tyrosine Alkaptonuria Phenylketonuria of phenylalanine Hyperalaninemia Leucinosis or maple syrup urine disease – of branched-chain a.a homocystinuria – of methionine Nonketotic hyperglycinemia – of glycine

117 PROTEIN CATABOLISM Has various indication:
Is carried out via proteolysis is the directed degradation (digestion) of proteins by cellular enzymes called proteases (various kinds) releasing peptide/A.A The digestion of proteins from foods as a source of amino acids (aas) The aas constituting “aa pool” are metabolized further (refer to aa catabolism)

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119 glutaminase liver NH+4 Non-liver cell glutamine NH+4 + α-ketoglu
Muscle-cell aa1 + α-ketoglu Aa aminotransferase glutamate Ala.aminotransferase + pyrv blood alanine Ala.aminotransferase + α-ketoglu glutaminase liver Oxidatv deamintn/GD glutamine glutamate NH+4 NH+4 + α-ketoglu glutamine Non-liver cell Glutamate +ATP NH+4 + α-ketoglu Glutmine synthetase Non-redtv amination Oxidatv deamintn/GD glutamate

120 One Carbon Metabolism . source of Diagram on next slide

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