Presentation on theme: "SFA 2073 Topic II Amino Acid & Proteins"— Presentation transcript:
1 SFA 2073 Topic II Amino Acid & Proteins Nik Norma Nik Mahmood (PhD)Faculty Science & TechnologyUni.Science Islam MalaysiaNILAI, N.Sembilan
2 OBJECTIVESTo Classify amino acids according to their structures and properties.To explain the meaning of pKa and pI of amino acidsTo understand the biochemical benefit of amino acidsTo describe the importance of some amino acids in the synthesis of important compoundsTo understand the biochemical benefit of proteinsTo 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 diseaseAmino 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 molalityApply the units of concentration used in medicine (g%, mmol, g/dl, IU/I etc.)Explain dilution, concentrated, saturated and supersaturated solutionExplain 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-1M =moles of solute (mol)Volume of solution (dm3 or liter)moldm-3molL-1m =moles of solute (mol)mass of solvent (kg)
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 multiplyby 100.eg 20g NaCl in 100 g salt solution20 x 100 = 20 % sodium chloride solution100b) mmol: millimol = 1X 10-3 mol or 1 mol= 103 millimolc) g/dl : gdl-1 = g in 1 deciliter solution10 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 IUInsulin: 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-caroteneVitamin C: 1 IU is 50 μg L-ascorbic acidVitamin D: 1 IU is the biological equivalent of μg cholecalciferol/ergocalciferolVitamin 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 solutionhas less amount of water and more amount of the substance. Forexample concentrated H2SO4 has 2% water and 98% H2SO4 anddilute has less amount of substance and more amount of waterc) saturated solutioncontains the maximum amount of a solute that will dissolve in a given solvent at a specific temperature.d) supersaturated solutioncontains more solute than is present in a saturated solution at a specific temperature.e) biological solution: concentration is described as hypertonic or hypotonicHypertonic 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.
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 KapKb:The negative logarithm of the base protonation constant Kbthe 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 pKaTitration of 100 mL 0.1 M CH3CO2H with 50 mL 0.1M NaOHCH3CO2H + NaOH CH3CO2‾+Na +H2Ostoichiometric coefficient 1:1Initial mole CH3CO2H = 0.01Final mole CH3CO2ˉ =0.005Unreacted CH3CO2H = = 0.005pH value can be determined by using pH meterSubstituting all the values in the equation, can get pKaBy 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 proteinProteinnPeptide bondsDifferent coloured balls & box => Amino Acids
16 Amino Acid: Structure & Function Amino acid (a.a) 20 altogether = std aaall aa share a general formula R-CH-NH21 aa differ from the other by the feature of –RClassified 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 aaCOOH
17 Aliphatic Non-polar Amino Acid hydrophobicityProperties: glycine and alanine are also found in the free form.
18 Aromatic Amino AcidProperties: tryptophane phenylalanineAre 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’sAre very hydrophilicimidazole 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 bodyEssentialNonessentialIsoleucineAlanineLeucineAsparagineLysineAspartateMethionineCysteine*PhenylalanineGlutamateThreonineGlutamine*TryptophanGlycine*ValineProline*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 zwitterionicCan 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 carboxylicgroup 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 noelectrical 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 currentiii) the molecule has minimum solubility.iv) Ip of all aa lie in the range of pHIsoelectric pH of an aa solution is given by:pH = ½ (pK1 + pK2)CH3-CHCOOHCH3CH COO¯NH2N+H3Neutral un-chargedNOT THISZwitterion. Neutral but chargeHigh pH regionLow pH region
25 For aliphatic aa50% as cationic 50% as zwitterion50% as anionic 50% as zwitterionE.gThe 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 moleculesChemical properties: involve –COOH & involve –NH2i) involve –COOH• decarboxylation or formation of amine & CO2eg. histadine histamine + CO2tyrosine tyromine CO2tryptophan tryptamine + CO2lysine cadaverine CO2Glutamic 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 proteinsAmide formation (at 2nd —COOH)aspartic + NH asparagineglutamic + NH glutamine (than N donated for N.Asynthesis)ii) involve –NH2:● formation of carbamino compound–NH2 + CO –NH-CO2Heg transport of CO2 by hemoglobin from tissue to lungHb–NH Hb–NH-CO2H(carbamino-Hb)● Transamination eg in metabolism pathwayRCHCOOH + R’CCOOH RCCOOH + R’CCOOHNH O O NH2● oxidative Deamination eg. in metabolism pathwayRCHCOOH RCCOOH NH3NH 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 Transmethylationmethyl group of methionine may be transferred to an acceptor to become intermediates in metabolic pathwayFormation of S-S bridge.sulfhydryl (-SH) group of cysteine can form the S-S bondwith another cysteine residue intrachain or interchain2 cysteines cystineFunction 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 waterb) 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 reactionsTyrosine thyroxine, a thyroid hormoneSerine cycloserine an anti-tuberculous;azaserine, an anti-cancer moleculeArginine ornithine and citrulline, intermediates in urea cycle
31 2. Structure and function of proteins To enable to:Describe the formation of peptide bondsDescribe the four levels of protein organization with reference to primary, secondary, tertiary and quaternary structure of proteins using haemoglobin as exampleExplain how structure of protein determines its function by looking at examplesDifferentiate between globular and structural proteins with examples eg immunoglobulin, hemoglobin, collagen, keratin etcDescribe the functions of proteinRelationship between structural protein and its function in health and disease.
32 i- starch maltose + shorter chain starch Proteins: Biological Functionsas biological catalysts of the chemical reactions that occur within the cell examples:i- starch maltose + shorter chain starchii- protein amino acids + peptide chainiii- triglyceride f f a + mono + diiv- ATP ADP +Piα-amylasetrypsinlipaseglyceridesphosphatase
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 organismeg peptide and protein hormones; allosteric enzyme; gene inducers & repressors.As transporter molecules eg. hemoglobin; GLUT,SGLUTi- hemoglobin transport O2 from tissue to lungs; myoglobin transport O2 intracellularii- 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―NO Hfeature 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 H2Othis 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
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 polypeptideThe residues are joined by peptide bondsChanges 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 bondC-terminalN-terminal
39 Effect of surrounding pH on the structure At neutral pHProtein with basic aa will have overall positive charge.Andthat with acidic aa will have overall negative charge
40 Effect of surrounding pH on the structure contEffect 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. Insuliniii) 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 directionβ- pleated sheets: the chains are folded so they lie alongside each otherH2 bondβ-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 solubilityresist 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 shapeis 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 ORWhen "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 arrangementThis 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
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 ormore parts.only exists, if there is more than one polypeptide chain present in a complex proteinHemoglobin: an oxygen carrying protein in red blood cells which is made of 4 parts.
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 ofI- Protein metabolism (synthesis and breakdown)II-Amino Acid metabolism (synthesis and breakdown)WILL PROCEED WITHProtein metabolism (synthesis and breakdown)
54 PROTEIN SYNTHESISproteins 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.atranscription : 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 tRNACOMPONENTS PRESENT IN THE PROCESSAminoacid carriedanticodoncodonRIBOSOMEmRNA
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 AA 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 TRANSLATIONEven though every protein begins with the Methionine amino acid, not allproteins will ultimately have methionine at one end. If the "start"methionine is not needed, it is removed before the new protein goes towork (either inside the cell or outside the cell, depending on the type ofprotein synthesized)
61 TRANSLATION PEPTIDIC BOND IS FORMED 2º AMINOACID: Glycine (only in this case) in site A.APEPTIDIC 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 AbsorptionIs carried out via proteolysisis the directed degradation (digestion) of proteins by cellular enzymes called proteases (various kinds) releasing peptide/A.AThe digestion of proteins from foods as a source of amino acids (aas)The aas constituting “aa pool” are metabolized further ( aa catabolism)
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 beansIn 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 juiceIn 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 + peptonesCasein(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)ChymortypsinCarboxy peptidases( 2 types: A and B) are exopeptidases;splits one amino acid at a time fr free end.Elastases : a serine proteaseCollagenases act on protein present in collagen/connective tissue yielding peptide- Jejunum-ileumFood remnant meet intestinal juice.Enzymes in intestinal juices:Amino peptidase: peptides tripeptidesEnteropeptidase/EnterokinaseProlidase: acts at terminal prolineDi and tri-peptidase: Di and tri-peptide amino acids
67 Absorption Of Amino Acids Absorption is by active transportSite of absorption is- ileum and distal jejunum: amino acids- duodenum and proximal jejunum: di and tri-peptidesAfter absorption, amino acids and di and tri-peptides (if any) are carried by portal blood to liver, partly :i- are taken up by liver cellsii- 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 lossii- cell waste amnt of aa taken-up < amnt of aa lossiii- cell grows amnt of aa taken-up > amnt of aa lossRegulatory of Amino AcidIf 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 proteinDietary proteinenzymic proteolysise.g trypsin/pepsinOUTSIDE CELLINSIDE CELLA A pool─ C skeletonsynthesis─NH2proteinPrecursors for other moleculesExcreted as urea & uric acidenergyNew AAN containing moleculesHealthy & in young subject >> aa breakdown
71 Amino acid synthesisis 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 mediaI –NH2 group is incorporated into α-keto acid through 2 types of reactions:i- non-reductive transaminationi) glutamate/aspartate as –NH2 donorii) glutamine/asparagine as –NH2 donoriii) branch chain aa as –NH2 donorii- reductive transaminationII- 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 regularReaction is catalysed by i) enzyme aminotransferase or transaminase ;ii) required co-enzyme pyridoxal-5’-phosphate (PLP)OR1—C—C--O‾ R1—CH—C--O‾ α-ketoglutarateO O NH3Acceptor α-ketoacidNew AAreferred as pair
74 iii- asparagine and an α-keto acid (pyruvate) COOˉCH3COOˉCH3+ CH-NH2COOˉCO-CH2-CH C=ONH NH COOCOO-CH2-CHNH3+PyruvateAspatateAsparagineAlanineTransaminase** 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)COOCOOCOOCOOOOC-CH2-CH2-CH + (CH3)2CH-CH O=C-CH2-CH2-CH + (CH3)2-CH-CNH NH NH NH OGlutamate valine glutamine BC α-oxoacid
76 Enzyme Transaminase/Aminotransferase requires co-enzyme pyridoxal-5’-PO4 , abbreviated (PLP).a derivative of vitamin B6R 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].
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 donorThe enzyme GOGAT is NADPH dependentglutamine + 2-oxoglutarate + NADPH + H+ ---> 2 glutamate+ NADP+GOGATGOGAT: enzyme glutamine oxoglutarate amidotransferase
81 II- Incoporation of NH4+ ion: i) Reductive aminationreaction of α-ketoglutarate with NH4+ leading to formation of glutamate (in mitochondria & cytoplasm).α-ketoglutarate is –NH2 acceptorcatalysed by glutamate dehydrogenase, the enzyme is NADH dependentreaction 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 presentNH4+ is from oxidative deamination of glutamate (in extrahepatic tissue)
82 Reductive Amination : left - right GD+ NH4+ + NADH + H++ NAD+ + H2OEnzyme: Glutamate DehydrogenaseNH3 + 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 & intestineThis 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- CHCOO-CH2-CH2- CH+ ATP + NH4++ ADPNH2NH3++ PiNH3+GlutamineGlutamateGlutamine Synthetase (GS)COO-COO-CO-CH2-CHNH NH3+COO-CH2- CH+ ATP + NH4++ ADP+ PiNH3+Asparagine SynthetaseAsparagineAspartate
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.
87 C skeleton of all non-essential aa are derivatives of: Glycerate -3-phosphatePyruvateΑ-ketogluterateOxaloacetateBut Tyrosine from essential aa phenylalanineOn basis of common precursor Ξ similarities in their syntheticPathway, aa can be grouped into 5 families.
88 glutamate family= synthesis of glutamate, glutamine, arg, pro. - C skeleton derive fr α-ketoglutarateserine family = synthesis of serine, glycine, cystein- C skeleton derive fr glycerate-3-phosphateaspartate family = synthesis of aspartate, lysine, methionine, asparagine, threonine- C skeleton derive fr oxaloacetatepyruvate family = synthesis of alanine, valine, leucine, isoleucine- C skeleton derive fr pyruvatearomatic family = synthesis of *phenylalanine, tyrosine, *tryptophan *EAA
89 Glutamate family- key substrate is α-ketoglutarate fr TCAGlutamate is produced by GD and is the principle rxn of fixation of NH3 in bactriaglutamine is produced by ATP-requiring +n of NH3 to glu and the rxn fnc as a major means of assimilating of NH3 fr environmentRegulation 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 moleculesUtilization 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 compoundsDecarboxylation- one carbon metabolism
92 glutamate + NAD+ −− NH+4 + α-ketoglutarate Removal of amino groupOccurs by- transamination- oxidative deamination (only happens with glutamate ) catalyses by glutamate dehydrogenaseglutamate + NAD+ −− NH α-ketoglutarateTransamination ( 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
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-ketoglutaratehappens only with glutamatecatalyses 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 deaminationglutamateNew α-keto acidalanineAlanine transaminationtransaminationexcess aapyruvateα-ketoglutaratealanineglutamateGDNH4++ NADHH2O + NAD+LiverAlanine transaminationTo urea cycleα-ketoglutaratepyruvateα-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 GDThe 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 cytosolMain substrates: NH3, CO2 and Aspartate.In the matrix of mitochondria occurs CPS I and OTC catalysed rxn,CPS rxn uses 2ATP and reaction is irreversibleCitrulline Ornithine occur in cytosol, in 4 steps-Citrulline is tranported across the inner membraneby a carrier neutral aa.- enzymes are arginosuccinate synthase, arginosuccinate lyase and arginaseUrea transferred to kidney through blood and excreted as urine
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 basesWhy the synthesis occurs?These molecules are synthesized because they are important to the body.The synthesis process
108 serotoninFncn to influence the functioning of the cardiovascular, renal, immune, and gastrointestinal systemsAny 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 : melatoninCarrier : carnithineAs hormone: tyroxine,Dilating/constriction of blood vessel: histamineExhibit 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 glucogenicThose that yield acetyl-CoA are divided into 2 groups.a) Those that yield pyruvate as intermediate: Ala, Cys, Gly, Ser and Thrb) Those that do not yield pyruvate as intermediate: Phe, Lys, Leu Trp and Tyr
113 Decarboxylation of amino acid is effected by decarboxylase enzyme, PLP dependentProducts are alkylamine + CO2 . The alkylamine are neurotransmittersThere 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) HDCHOOC-CH2-CH2-CH(NH2)-COOH ───→ CO2 + HOOC-CH2-CH2-CH2NH2GABA is a neurotransmitter in brainGAD(GABA)Aromatic L-aa decarboxylase synonyms to DOPA decarboxylase, tryptophan decarboxylase, 5-hydroxytryptophan decarboxylase, AAAD.tryptophan ───→ tryptamine + CO2Tryp D
115 A.As Metabolic Disorder Diseases Are diseases resulted from disorders of a.as processing/metabolism due toInherited/genetic defects that cause deficiency of certain enzymes fori) the breakdown of amino acids orii) the body's ability to get the amino acids into cells oriii) Amino acid TransportSymptoms 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 albinismTyrosinemia of tyrosineAlkaptonuriaPhenylketonuria of phenylalanineHyperalaninemiaLeucinosis or maple syrup urine disease – of branched-chain a.ahomocystinuria – of methionineNonketotic hyperglycinemia – of glycine
117 PROTEIN CATABOLISM Has various indication: Is carried out via proteolysisis the directed degradation (digestion) of proteins by cellular enzymes called proteases (various kinds) releasing peptide/A.AThe digestion of proteins from foods as a source of amino acids (aas)The aas constituting “aa pool” are metabolized further (refer to aa catabolism)