Protein Part I Summary PDF

Proteins 040817231 (Introductory Biochemistry) By: Dalia Shaaban, PhD Lecturer of Biochemistry, Faculty of Science, Alexandria University  Proteins The most abundant macromolecules in living cells. In 1838, Derived from t...

Proteins 040817231 (Introductory Biochemistry) By: Dalia Shaaban, PhD Lecturer of Biochemistry, Faculty of Science, Alexandria University  Proteins The most abundant macromolecules in living cells. In 1838, Derived from the Greek word “ PROTOS” = Primary or holding first place, indicating that protein is the most important cell constituents (cytoplasm & cell membrane) & involved in each function. Nitrogenous organic compounds composed of the basic elements :C, H, O, N. Complex macromolecules of the basic monomers (L- α-amino acids) linked together by strong covalent peptide bond. Proteins Each protein has specific & unique sequence of amino acids. For each protein, its amino acid content & sequence determines: ✓ Three dimensional (3 D) structure ✓ Biological function. About 300 amino acids are present naturally, but only 21 amino acids are involved in the formation of proteins (called primary, proteogenic, standard, basic). The rest amino acids are non-standard (not- proteogenic). Standard Amino Acids Proteogenic a.as: coded: naturally encoded in the genome of organisms for the assembly of proteins. In eukaryotes: they are 21 Standard (primary, basic) a.as = 20 a.as (standard genetic code) + selenocysteine (incorporated by special translation mechanisms). Pyrrolysine sometimes considered “the 22nd a.a ”, is not listed here as it is not used by humans. Each a.a has a name, as well as a 3 letter or single letter mnemonic code. All 21 a.as are biologically essential, their deficiency impairs protein synthesis.  Standard Amino Acids α-Form: as the amino group (-NH2) is attached to C atom next to the carboxyl group (-COOH). For all the common amino acids except glycine, the α-C is bonded to 4 different groups: a carboxyl group (α-COOH gp) an amino group (α- NH2 gp) a hydrogen atom (H) a side chain (R gp) But in glycine, the R group is another hydrogen atom. All have free α- NH2 & α-COOH groups. L- configuration: according to their optical activity (levorotatory counterclockwise behavior). Standard Amino Acids They form peptide bond in peptides & proteins Condensation (dehydration) Peptide bond is a covalent chemical bond formed by linking the carboxyl group of one free a.a molecule to the amino group of another.  Standard Amino Acids Amino acids have different R (side chain), that responsible of different properties: Basic/charged(+) Shape Size Charge Neutral H-bonding capacity Hydrophobic characters Acidic/charged(-) Chemical reactivity.  Standard Amino Acids Amino acids are classified based on: Chemical nature in solution ( neutral, acidic, basic). Chemical structure of R ( Aliphatic, dicarboxylic, diamino, OH, S-containing, aromatic, imino “heterocyclic”. Polarity of R side chain ( hydrophilic, hydrophobic). Nutritional requirements (nutritionally essential & nonessential). Classification of Standard Amino Acids ( according to polarity of R) Polarity of a.a and its water solubility properties are mainly influenced by: its polar amino and carboxyl groups Side chain structure i.Non polar R ( HY DR OPHOB IC) R are either aliphatic ( branched & linear) aromatic. Glycine is the simplest a.a. Polarity decreases with increasing the hydrocarbon chain length. Proline is an imino acid (α- NH2 & its side chain form a ring structure, Heterocyclic, so imino –NH gp rather than NH2 gp.  Classification of Standard Amino Acids ( according to polarity of R) i. Non polar R ( HY DR OPHOB IC) Methionine are S- containing a. a ( Thioether). Phenylalanine contains a phenyl ring. Tryptophane has indole ring ( 2 fused rings & an NH gp). Tyrosine is aromatic nonpolar Classification of Standard Amino Acids ( according to polarity of R) ii.Polar R (HYDROPHYLIC) 1) Uncharged A.As R is uncharged side chain has either O, S , or N atoms, enabling them to form H bonds with water. Serine & Threonine have OH gp. in R. Cysteine has –SH (sulfhydryl gp) in R Asparagine & Glutamine with Amide gp in R.  Classification of Standard Amino Acids ( according to polarity of R) ii.Polar R (HYDROPHYLIC) 2) Charged A.As A. Negatively Charged: dicarboxylic a.as Aspartic & Glutamic acids with COOH gp in R, deprotonated (COO-): -ve charge R at physiological pH. B. Positively Charged: diamino a.as Lysine & Arginine: long R terminated by + vely charged NH2 & guanidium groups respectively, at physiological pH. Histidine with imidazole group, + vely charged at physiological pH. Classification of Standard Amino Acids, based in nutritional requirements All 21 a.as are biologically essential. Humans can synthesize 12 (nutritionally Non-Essential a.as) from each other or from other molecules of intermediary metabolism. While 9 a.as nutritionally Essential a.as as they are cannot be synthesized in human body and must be consumed (usually as their protein derivatives).  Ionization of Standard Amino Acids In most of body fluid, amino & carboxlic groups are ionized. At physiological pH (pH 7.4), Monoamino monocarboxylic acids exist predominantly as zwitterions (Z.I). Zwitterions: dipolar ions, the ionization state of an a.a, in a solution, containing equal numbers of +ve & -ve charges due to: deprotonation of COOH gp (–vely charged: COO-) protonation of NH2 gp (+ vely charged: NH3+). Unionized form of a.a H2O Z.I is electrically neutral, of net charge= zero. Ionized form Amphoteric molecule as they can act as acid or base. pKa1 pKa2  Ionization of Standard Amino Acids The ionization state of an a.a varies with pH. In acidic solution, Cation form of a.a: the amino gp is protonated (NH3+), carboxylic gp is protonated (COOH). As pH is raised, COOH gives up a proton and turns to COO-, forming dipolar Z.I. At higher pH ( > 9), Anion form of a.a: the protonated amino gp loses a proton (-NH2). Isoelectric point pI: pH at which the amino acid exists in Z.I. Therefore Z.I does not move in an electric field At pI, the a.a is precipitated, has the lowest solubility Each side chain has its own pKa value for its ionization. Importance of Standard Amino Acids 1. Formation of proteins: a.as are joined by peptide bonds to form peptides & proteins. 2. Formation of glucose: glucogenic a.as are converted to glucose in body. 3. Enzyme activity: the thiol (-SH) of cysteine has an important role in certain enzyme activity. 4. Transport & storage of amino N in the form of ammonia. 5. Free a.a can act as buffer, due to amphoteric character. Histidine the best buffer at physiological pH. Importance of Standard Amino Acids 6. Formation of biologically important compounds a. Glycine involved in bile salts, cysteine converts toxins to less toxic compounds that have detoxification activity. b. Glutamic acid, cysteine & glycine form Glutathione: important Antioxidant. c. Aspartic & glutamic acids are precursors of pyrimidine base ( cytosine, thymine, uracil). d. Glycine, aspartic & glutamine are precursors of purine bases ( adenine & guanine). e. Hormones synthesis ( ex: adrenaline, thyroxine, melanin derived from tyrosine )  Peptides &Proteins ▪ Dipeptides: polymer of 2 a.as (linked by one peptide bond)(Aspartam). ▪ Oligopeptides: (3-10 a.as) ▶Tripeptide (3 a.as linked by 2 peptide bonds), ▶Tetrapeptide (4 a.as linked by 3 peptide bonds), ▶ Pentapeptide (5 a.as linked by 4 peptide bonds). ▪ Polypeptides: (11-100 a.as), Molecular weights ˂10,000 Dalton. ▪ Proteins: (˃ 100 a.as) ▶ One or more polypeptide chains. ▶ Higher molecular weights ˃ 10,000 Dalton.  Biologically Important Peptides Peptide a.as in peptide Function Insulin 2 chains of 30 & 21 Regulates BGL Glucagon 29 Regulates BGL Oxytocin 9 Stimulates uterine contraction Vasopressin 9 Increase BP & antidiuretic Bradykinin 9 Powerful vasodilator stimulate pain receptors Glutathione 3 Power antioxidant & detoxify H2O2  Levels of ProteinStructure ▶ There are four levels of protein structure: I. Primary Structure II. Secondary Structure III. Tertiary Structure IV. Quaternary Structure  Levels of ProteinStructure I. Primary (1o) Structure ▪ It refers to it’s a.a sequence (ORDER), which are joined together by peptide bonds (amide bonds) between the carboxyl & amino groups of adjacent a.as. ▪The backbone of all proteins consists of a repetitive unit [-N-Cα(R)-C(O)-]. ▪Only the R-group side-chains vary.  Levels of ProteinStructure II.Secondary (2o) Structure ▪ It refers to folded structures within a polypeptide in α-Helix & β-Pleated Sheet. ▪ Hydrogen bonds, which form between: the carbonyl O of one a.a backbone & the amino H of another, held the shape of the secondary structures. ▪ These interactions do not involve R group atoms.  Levels of ProteinStructure III.Tertiary (3o) Structure ▪ It refers to the folded 3D shape of a protein. ▪ Known as the native structure or active conformation as it is very important for protein function. ▪ The proteins are folded to place the most hydrophobic a.a in the interior (out of contact with water) and hydrophilic a.as in the exterior. ▪ The secondary structures (α-Helices & β- Sheets) fold based on interactions, between R-groups of a.as, including: Non- Covalent: hydrophobic, ionic, hydrogen, van de Waals Covalent: disulphide bridge.  Levels of ProteinStructure VI. Quaternary (4o) Structure ▪ It refers to the association of polypeptide chains. ▪ Sometime proteins have inorganic prosthetic group. ▪ Some proteins are oligomeric i.e, contain multiple polypeptide chains (multi subunits). ▪ These proteins are functional when all its subunits are present. ▪ The simplest sort is a dimer, consisting of two identical subunits (homodimer). ▪ Hydrogen, ionic, hydrophobic interactions & disulphide bridge also hold the subunits together to give quaternary structure.  Levels of ProteinStructure VI. Quaternary Structure ▪ Hemoglobin Hb in RBCs, is Carrier of O2 all over the body. ▪ Composed of: 4 polypeptide chains ( 2 α & 2 β chains): tetrameric protein. 4 inorganic prosthetic groups (haem/heme group: contains Fe+2 which attract O2). ▪ Each Hb molecule carries 4 O2 molecules= 8 O atoms. ▪ Heme is responsible for blood color, ✓ oxygenated Hb isred ✓ Unoxygenated Hb is purple  Structure-function Relationship ▪ The three-dimensional structural conformation of protein provides & maintains its functional characteristics for a native protein. ▪ Native conformation of protein is its natural biological conformation. ▪ The three-dimensional structure is dependent on the 1o structure. So, any difference in 1 o structure may produce a protein which cannot serve its function.  Structure-function Relationship ▪ Even a single a.a substitution alters the structure & thereby the function. ▪ In sickle cell anemia (HbS), glutamic acid in β-chain of Hb is replaced by valine. ▪ Less soluble Hb, increase clot. ▪ Inefficient for O2 delivery ▪ Lower RBCs live (20 days, compared to 120 for normal). ▪ Bone marrow cannot replace fast enough. ▪ Decrease RBCs count……..Anemia  Classification of Proteins, based on Composition &Solubility i. Simple Proteins: contain only amino acids. ii. Conjugated Proteins:a non-protein part (prosthetic group) is combined to the protein iii. Derived Proteins: A. Primary: derived by processes causing slight changes in native protein as coagulated proteins B. Secondary: derived by progressive hydrolysis of protein results in smaller chains as peptones & peptides  Classification of Proteins, based on shape 1. Globular Proteins Forming ball like, more compact and rounded shape water soluble, so in water environment: cell, tissue fluid, blood, lymph. Contain several types of secondary structure. Functional & Metabolic role as: catalysts, transport & regulation as: enzymes, antibodies, plasma proteins, insulin, hemoglobin. 2. Fibrous Proteins Forming long fibers, highly organized Due to large no. of hydrophobic R groups, water-insoluble stable structure. Contain single type of secondary structure. Structural role for support & protection: as collagen in bone & hair  Classification of Proteins, based on Nutritional value Nutritional value is determined by presence of essential a.as. 1. Complete (firstclass) proteins ✓ all essential a.as are present in proper portions. ✓ Good for heath. ✓ Animal source : milk, egg, meat, fish 2. Incomplete proteins ✓ Lack one or more essential a.as ✓ Unable to support health & growth completely. ✓ Plant source: cereals, bean, nuts, gelatin 3. Partially incomplete proteins ✓ all essential a.as are present BUT in improper portions to the metabolic need of organism. ✓ source: wheat, rice, corn.  Classification of Proteins, based on Functions ▪ Catalytic proteins: enzymes ▪ Structural proteins: collagen, elastin ▪ Growth & maintenance of tissues. ▪ Contractile or motile proteins: myosin, ▪ Maintain proper pH & balanced fluids. actin. ▪ Valuable energy source in excessive exercise or ▪ Genetic proteins:histones. inadequate calorie intake. ▪ Receptors & membrane transport proteins. ▪ Transport proteins:. hemoglobin, myoglobin, albumin, transferrin. ▪ Regulatory proteins (hormones) & messengers: ACTH, insulin, growth hormone ▪ Protective & defense proteins: immunoglobulins, interferons, clotting factors. ▪ Storage or Nutrient proteins: egg albumin & milk casein. ▪ Toxic proteins: snake venom.  Properties of Proteins Proteins exist as colloidal particles ( 5-100 μm), heavier than water. The colloidal protein solutions generate osmotic pressure, that depends on protein conc., but inversely proportional to its Mwt. Oncotic pressure is the osmotic pressure induced by proteins [as albumin in plasma], balancing fluids in body. Macromolecules with high Molecular weights with wide variations. Insulin (5,700); Hemoglobin, Albumin (69,000); Immunoglobulins (1,50,000). ▪ Solubility: Globular proteins are more soluble than fiberous. Smaller molecules are more soluble than larger. Shape of the proteins also vary. Insulin is globular, Albumin is oval in shape, Fibrinogen molecule is elongated.  Properties of Proteins Amphoteric nature & Isoelectric Point (pI) of protein Determined by it’s a.a composition. The a- NH2 & COOH groups are utilized for peptide bond formation (not ionizable). The pI of the protein is influenced by all other ionizable groups of R side chain present in the protein. At the pI value, the number of anions and cations present on the protein molecule will be equal and the net charge is zero. so, the proteins will not migrate in an electrical field. At the pI, solubility, buffering capacity will be minimum; and precipitation will be maximum. On the acidic side of pI, the proteins are cations and on alkaline side, they are anions in nature. The pI of pepsin is 1.1; human hemoglobin 6.7; lysozyme 11.  Precipitation of Proteins The stability of proteins in solution will depend mainly on the charge & hydration. Polar groups of the proteins tend to attract water molecules around them to produce a shell of hydration. Any factor, which neutralizesthe charge/removeswaterof hydrationwill therefore cause precipitation of proteins. As in the following procedures. 1. SaltingOut: Adding neutral salt (ammonium sulfate or sodium sulfate), to protein solution, removes the hydration shell and the protein is precipitated ( the protein is salted out). As a general rule, higher the Mwt of a protein, the salt required for precipitation is lesser. 2. IsoelectricPrecipitation as the Proteins are least soluble at their isoelectric pH. When milk is curdled, the casein forms the white curd, because lactic acid produced by the fermentation process lowers the pH to the isoelectric point of casein ( 4.6). 3. Precipitationby Organic Solventswhich influence dehydration & reduce the dielectric constant of the medium which also favors protein precipitation. Hence alcohol is a powerful protein precipitating agent, explaining the disinfectant effect of alcohol. 4. Precipitation by Heavy Metal Ions (Cu, Zn , Cd, Pb) & Alkaloidal Reagents (Trichloroacetic  Denaturationof Proteins ▪ Denaturation is rapturing the interactions (H, ionic, hydrophobic) those stabilize & maintain the protein conformation in space. ▪ There will be nonspecific alterations in 2o, 3o &4o structures of protein molecules. 1o structureis not altered during denaturation. ▪ The denatured protein solubility is decreased while its perceptibility is increased. It often causes loss of biological activity. ▪ Native proteins are often resistant to proteolytic enzymes, but denatured proteins will have more exposed sites for enzyme action ( cooked proteins are easily digested). ▪ Caused by: heating, treating with urea, salicylate, X-ray, UV rays, high pressure,vigorous shaking. ▪ Generally irreversible, but sometime it is reversible when the denatured proteins are re-natured with the removal of physical agent.  Digestion&Absorption of DietaryProteins ▪ Adult requires 70-100 g Protein/day. ▪ Source for: ✓ essential a.as ✓ a.as constituents for protein synthesis. ✓ C & N atoms used for synthesis of N- cellular & non N-containing metabolites. ▪ As large molecules, should be hydrolyzed by proteases to its a.as constituents to be absorbed by intestine.  Digestion &Absorption of DietaryProteins In mouth:mechanical breakdown by chewing, saliva aids in swallowing the partially mashed proteins through the esophagus into stomach. In stomach: Protein digestion takes a longer time than carbohydrate digestion, but a shorter time than fat digestion. Mechanical: muscular stomach contractions Chemical: gastric juices containing HCl & the enzyme pepsin,which initiate the breakdown of protein. ✓ Because of HCl, pH of stomach is acidic (1.5-2.5), killing microbes, denaturing & unfolding the dietary protein, activating pepsin from its inactive zymogen (pepsinogen). ✓ In the denatured proteins, the peptide bonds are more accessible for enzymatic digestion by pepsin. Pepsin begins breaking peptide bonds, creating shorter polypeptides.  Digestionof Dietary Proteins Inthe small intestine: the majority of protein digestion occurs, by: 1)Pancreatic juice: ✓ Bicarbonate neutralizes the acidic pH of chyme in intestine to 7. ✓ Pancreatic Proteases: Exopeptidases: cleave a.a\time from either carboxy/amino- terminal of peptide [carboxy/aminopeptidases] Endopeptidases:cleave internal peptide bonds producing small peptides[Trypsin, chymotrypsin]. Most of pancreatic proteases are secreted as inactive proenzymes [trypsinogen, chymotrypsinogen, , chymotrypsinogen, proelastase, procarboxypeptidases] 2) Intestinal enteropeptidases: secreted by the brush border of the small intestine, activating trypsin. ▪ Trypsinactivates the other protein-digesting enzymes (proteases). ▪ All together hydrolyze the internal peptide bonds, producing small peptides. 3) Intestinal proteases: aminopeptidases & dipeptidases, continue the enzymatic digestion.  Digestion&Absorption of DietaryProteins ▪ Tripeptides, dipeptides & individual a.as are final products of protein digestion, absorbed into blood stream. ▪ Proteins that aren't fully digested in the small intestine pass into the large intestine, excreted as feces. ❑ Tripeptides, dipeptides & single a.as enter the enterocytes of the small intestine using active transport systems, which require ATP. ❑ Once inside, the tripeptides and dipeptides are all broken down to single a.as which are absorbed into the bloodstream. ❑ Different L-a.as are transported by several types of active transport systems. ❑ D-a.as are transported by passive diffusion.  Metabolism of Proteins What happens to absorbed amino acids? ▪ the a.as are macronutrients (nutrients used in large amounts). ▪ the absorbed a.as are transported to the liver as checkpoint for a.a distribution & any further breakdown, which is very minimal. ▪ Dietary a.as become part of the body's a.a pool.  Metabolism of Proteins If the body has enough glucose& other sources of energy, a.as will be used in one of the following ways: ▪ Protein synthesis in cells around the body. ▪ Making nonessential a.as needed for protein synthesis. ▪ Making other N-containing compounds (porphyrins, carnitine, nucleotides) & precursors for signal molecules (neurotransmitters & hormones). ▪ Rearranged & stored as fat (there is no storage form of protein).  Metabolism of Proteins ▪ When caloric intake is inadequate. Glycogen & fat stores are low (starvation). Rigorous, continuous exercise (ultramarathon). Carbohydrate intake is inadequate. Protein is used for energy. ▪ a.a is used as energy source (a.a wasting). As last restore to give a.a Catabolism brain & RBCs glucose that metabolized as fuel, for an immediate source of ATP.  Metabolism of Proteins ▪ In order to use a.as to make ATP, glucose, or fat, a.a is catabolized (minimal pathway). 1) Deamination: removal of the a- amino by transaminases & deamination (oxidative/non-oxidative), in the liver & kidneys, released as toxic ammonia, transforms in liver into highly soluble urea which then transported to the kidneys and excreted excess N in the urine out of body. 2) Remaining C-skeleton of a.a is oxidized in TCA into CO2 & water & ATP  glycerol Glycerol-3p -ATP NADH DHAP NADH+ATP PEP ATP PYRUVATE