Introduction to Biochemistry & Proteins BIOC 210 Fall 2024-25 PDF

MOHAMMED AL-MANA COLLEGE FOR MEDICAL SCIENCES Introduction to Biochemistry & Proteins BIOC 210 Fall 2024-2025 CHAPTER OBJECTIVES Upon completion of this chapter, the student will be able to: - Classify the amino acids. - Draw the structure of amino acids...

MOHAMMED AL-MANA COLLEGE FOR MEDICAL SCIENCES Introduction to Biochemistry & Proteins BIOC 210 Fall 2024-2025 CHAPTER OBJECTIVES Upon completion of this chapter, the student will be able to: - Classify the amino acids. - Draw the structure of amino acids. - Draw the structure of peptide bond. - Differentiate the different levels of proteins. - Explain protein folding and denaturation - List the factors influence protein folding and denaturation. - Differentiate between myoglobin and hemoglobin structure. - Understand the concept of protein digestion.  What is Biochemistry? Biochemistry: also called biological chemistry, is the study of chemical processes in living organisms. Much of biochemistry deals with the structures and functions of cellular components such as biomolecules like proteins, carbohydrates, lipids, nucleic acids etc.  Why to study Biochemistry? Studying biochemistry provides in-depth knowledge in several key areas, including: 1. Chemical components of life. 2. Metabolism. 3. Genetics and molecular biology. 4. Enzymes and proteins. 5. Cellular processes. 6. Disease mechanisms.  Biomolecules All living organisms contain organic Macromolecule Building block macromolecules or biomolecules. (polymer) (monomer) These biomolecules are mainly made up of protein amino acids carbon, hydrogen, oxygen. carbohydrates monosaccharides They are also called "macromolecules“(Polymers) because they lipids glycerol + fatty acids are very large, containing long chains of carbon and hydrogen atoms and often nucleic acids nucleotides consists of repeating smaller molecules (Monomers) bonded together in a repeating pattern (to form polymers) Biomolecules  Practice 1 1. Explain how monomers are related to polymers. 2. Match the monomer on the left side to the macromolecule on the right side 1. Fatty acids & Glycerol B A. Protein 2. Monosaccharide B. Lipids 3. Nucleic acid 4. Amino acid T C. Carbohydrates D. Nucleotide 3. Match the polymer on the left to the macromolecule 1. DNA & A. Protein 2. Enzyme A B. Starch 3. Triglyceride * C. Nucleic acid 4. Polysaccharide B D. Lipids  Chapter 1: Proteins BIOC 210 Fall 2024-2025  Proteins Proteins are nitrogen containing most abundant organic macro-molecules widely distributed in animals and plants. Proteins are the fundamental structural components of our body. The Elements present in the protein are Carbon (C) Hydrogen (H2) Oxygen (O2) Nitrogen (N2) Small amounts of sulfur (S). Few proteins contain other elements such as Phosphorous (P), Iodine (I), Copper (Cu), Manganese (Mn), Zinc (Zn) and Iron (Fe) etc.,  Amino acids Amino acids are monomers ( subunit/ building block) of proteins. There are 20 different amino acids found in plants and animals They have amino and carboxyl group attached to the same carbon atoms The R group is often referred to as the side chain  The basic structure of all amino acids. Classification of Amino acids The amino acids are classified depending on the R groups as fallow: 1. Nonpolar R groups (side chain) Aliphatic R group Aromatic R group 2. Polar, uncharged R groups (side chain) 3. Polar, charged R groups (side chain)(Electrically charged side chains) Positively-charged (basic amino acids) Negatively-charged (acidic amino acids)  Non-polar R groups(side chains) Nine amino acids are classified as having non-polar side chains Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine, Proline, Phenylalanine, Tryptophan. Aliphatic R groups Aromatic R groups  Polar uncharged R groups (side chains) Those with Polar uncharged R groups have hydroxyl, amide, or thiol groups (sulfur). Six amino acids are commonly classified as having uncharged polar side chains. Serine, Cysteine, Asparagine, Glutamine, Threonine, Tyrosine.  Polar charged R groups(side chain) (Electrically charged side chains) 1. Basic amino acids They are Positively charged. These amino acids have two ─NH2 groups and one –COOH group. They are also called as Diaminomonocarboxylic acids Examples are Lysine, Arginine, Histidine  Polar charged R groups(side chain) (Electrically charged side chains) 2. Acidic amino acids They are Negatively charged. These amino acids have one ─NH2 groups and two –COOH group. They are also called as Monoaminodicarboxylic acids Examples are Aspartic acid and Glutamic acid Essential and non-essential amino acids Nutritionally, amino acids are of two types: Essential amino acids Non-essential amino acids These are the ones which are not These can be synthesized synthesized by the body and must be by the body and may not be the requisite taken in diet. components of the diet. valine, leucine, isoleucine, Alanine, Aspartate, Cysteine phenylalanine, and Glutamate……. threonine, tryptophan, methionine and lysine.  Features of proteins and properties BIOC 210 Fall 2024-2025  Physical properties of amino acids: Amino acids are colorless, crystalline solids All have a high melting point - They are soluble in water, slightly soluble in nonpolar solvents Aromatic amino acids can absorb UV light  General Properties of amino acids Amphoteric Nature The -NH2 and –COOH groups of amino acids act as proton donors (acids) or proton acceptors (bases). Due to this property amino acids are called amphoteric compounds. In neutral aqueous solutions the proton typically migrates from the carboxyl group to the amino group, leaving an ion with both a (+) and a (-) charge.  Zwitterions Zwitterions: The amino acid carries the positive and negative charges in equal number exist as dipolar ion or zwitterion. At this point the net charge on it is zero, that is positive charges and negative charges on the amino acid molecule equalize.  Isoelectric pH Isoelectric pH: The pH at which the amino acid exists as dipolar ion or zwitterion is called isoelectric pH.(pI).  Practice 2 1. For a molecule to behave like zwitterions what do you predict it must bear ? A. Uncharged groups B. Charged groups of opposite polarity C. Charged groups with same polarity 2. The isoelectric point of an amino acid is defined as …. A. The pH where the molecule carries no net electric charge. B. The pH where the carboxyl group is uncharged C. The pH where the amino group is uncharged  Peptide bond formation Proteins are composed of amino acids linked in a linear sequence by peptide bonds. The peptide bond is formed between the amino group of one amino acid and the carboxyl group of another amino acid with the removal a water molecule. Draw the structure of Dipeptide using the amino acids Glycine and Alanine 63  Practice 3 1. Draw the Zwitterion for the following amino acids. (a) Cysteine (b) Lysine (c) Aspartic acid 2. Draw the structure of a Dipeptide using the amino acids Asparagine and Glutamic acid. 3. Draw the structure of a Tripeptide using the amino acids Alanine, Cysteine and Glycine.  Classification of proteins Compounds of two amino acids linked by a peptide bond are know as dipeptides, Those with three amino acids are called tripeptides, and those with large number of amino acids (100) are called polypeptides. More than 100 amino acids it is called protein. CLASSIFICATION OF PROTEINS :- Proteins are classified based on 1. Structure and shape 2. Biological functions 3. Composition  Classification of proteins 1. Structure and shape: a) Fibrous proteins :- They are insoluble and consist of long parallel chains cross-linked at many points along their length. They are structural proteins. Ex :- Keratin found in hairs, nails, hooves, horns etc. Collagen found in muscles b) Globular proteins :- The polypeptide chains are tightly folded to form a spherical shape. They are soluble proteins. They are functional proteins Ex :- Myoglobin, Haemoglobin  Practice 4 Compare Fibrous Proteins & Globular Proteins Fibrous Proteins Globular Proteins  Classification of proteins 2. Biological functions 1. Defence Proteins : These proteins defend against other organisms. Example : immunoglobulins (antibodies) 2. Contractile or Motor proteins : Proteins of skeletal muscle are involved in muscle contraction and relaxation. Example : Actin 3. Transport or Carrier proteins: These proteins help in transport of ions or molecules in the body. Example: Hemoglobin 4. Storage proteins : These proteins provide nutrition to growing embryos and store ions. Example: Egg albumin 5. Enzymatic proteins : Enzymes catalyze a variety of reactions Example: Lipase 6. Regulatory or hormonal proteins: They regulate metabolic activities. Example: insulin 7. Structural proteins : These proteins help in strengthening protecting biological structures. Example : Collagen 8. Receptor proteins : These proteins recognize and adhere to the ligand molecules on the cell surface or in the cytoplasm. Example: Insulin receptors  Classification of proteins 3. Composition Based on the solubility and physical properties the proteins are classified into 3 different classes: a) Simple proteins : These are proteins which on complete hydrolysis yield only amino acids. Ex : Protamines, Histones, Albumins, Globulins b) Conjugated proteins : These are proteins which in addition to amino acids contain a non-protein group called prosthetic group in their structure Ex : Nucleoproteins, Glycoproteins, Lipoproteins, Chromoproteins, Haemoproteins, metalloproteins c) Derived proteins : These are proteins formed from native protein by the action of heat, physical forces or chemical factors. Ex : Proteoses, Peptones  Practice 5 Name the non-protein part or prosthetic group in the following conjugated proteins: 1. Nucleoproteins 2. Glycoproteins 3. Lipoproteins 4. Chromoproteins 5. Haemoproteins, 6. Metalloproteins  Levels of Protein structure Protein Structure 1. Primary - Sequence of amino acid. 2. Secondary - Interaction of amino acids close in primary sequence. 3. Tertiary - Interaction of amino acids distantly located. 4. Quaternary - Interaction of protein subunits  Primary structure Primary structure is the linear sequence of amino acids held together by peptide bonds in its peptide chain. The peptide bonds form the backbone, and side chains of amino acid residues project outside the peptide backbone. The free –NH2 group of the terminal amino acids is called N-terminal end and the free – COOH end is called C- terminal end.  Primary structure The amino acids are counted from the N-terminal as amino acid 1 towards the C- terminal end Presence of specific amino acids at a specific number is very significant for particular function of a protein. Any change in the sequence is abnormal and may affect the function and properties of protein. Example of Primary structure of Protein is Insulin  Primary structure A single amino acid change from glutamic acid to valine results in the disease called sickle cell anemia, an inherited disease that affects red blood cells. Sickled Cells  Secondary structure In Secondary structure the polypeptide chains may become folded or twisted in various ways. The most common ways are : α - helix β - pleated sheet These forms are referred to as the secondary structure of the protein. The linkages or bond involved in the secondary structure formation are Hydrogen bonds and Disulphide bonds.  Secondary structure α - helix : One polypeptide chain forms regular helical coils called a α- helix. These coils are stabilized by hydrogen bonds between carbonyl oxygen and amide hydrogen. (C=O….H-N) Thus in α- helix intrachain hydrogen bonding is present. The helices can be either right-handed or left-handed. Ex:-The proteins of hair, nail, skin contain a group of proteins called keratins rich in α-helical structure.  Secondary structure β - pleated sheet : 2 different polypeptide chains become linked in parallel flat sheets Sometimes the chains run in opposite directions called Antiparallel. The hydrogen bonds are formed between the carbonyl oxygens and amide hydrogens of the adjacent extended polypeptide chains. Thus, the hydrogen bonding in β - pleated sheet structure is interchain. The structure is slightly pleated due to the angles of bonds.Ex :- Silk fibroin, a protein of silkworm is rich in β - pleated sheet  Secondary structure α - helix β - pleated sheet intrachain hydrogen bonding interchain hydrogen bonding Parallel to peptide bond Perpendicular to peptide bond Hydrogen bond  Practice 6 Compare α - helix & β - pleated sheets α - helix β - pleated sheets  Te r t i a r y S t r u c t u r e Protein tertiary structure is the three-dimensional shape of a protein. The tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains. Amino acid side chains may interact and bond in number of ways.  Te r t i a r y S t r u c t u r e There are four ways in which parts of the amino acid chains interact to hold the 3d tertiary shape together : 1. Disulphide bonding (disulfide bridges) formed when two cysteine molecules combine in which –SH groups are oxidized. 2. Hydrogen bonding between polar groups on the side chain. 3. Ionic bonds formed between –NH2 and –COOH groups 4. Hydrophobic interactions. (Normally occur between non- polar side chains of amino acids which repel water). Ex : Example of tertiary structure is the enzyme lipase.  Quaternary structure The quaternary structure of a protein is the association of several protein chains or subunits into a closely packed arrangement. Each of the subunits has its own primary, secondary, and tertiary structure. The subunits are held together by hydrogen bonds and van der Waals forces between nonpolar side chains. Quaternary structure is maintained by the same forces which are active in maintaining the tertiary structure Ex: Haemoglobin  Practice 7 Match the following 1 Interchain hydrogen bonding D A Primary structure 2 alpha helices and beta sheets B B Secondary structure 3 The simplest level of protein C Quaternary structure structure A 4 a molecule or ion that can act both as D β - pleated sheet an acid and as a base. C  Protein folding The amino-acid sequence (or primary structure) of a protein defines its native conformation. A protein molecule folds spontaneously during or after synthesis. While these macromolecules may be regarded as "folding themselves”. The process also depends on: 1. solvent 2. the concentration of salts 3. the temperature and 4. the presence of molecular chaperones.  Protein Folding Disruption of the native state or denaturation of proteins Under some conditions proteins will not fold into their biochemically functional forms. Temperatures above or below the range that cells tend to live in will cause thermally unstable proteins to unfold or "denature" (this is why boiling makes an egg white turn opaque).  Protein Folding Denaturing of proteins can be caused by 1. High concentrations of solutes 2. Extremes of pH 3. Mechanical forces 4. The presence of chemical denaturants 5. High temperature  Protein Folding A fully denatured protein lacks both tertiary and secondary structure and exists as a so-called random coil. Under certain conditions some proteins can refold; however, in many cases denaturation is irreversible. Chaperones or heat shock proteins: Enzymes that are assist other proteins both in folding and in remaining folded. Diseases result from wrong protein folding: Alzheimers disease, Cystic fibrosis, Mad Cow disease, An inherited form of emphysema. Even many cancers.  Practice 8 1. Which of the following is an example of protein denaturation? (a) protein binds with a substrate, lowering the activation energy of a reaction (b) Several amino acids are joined together via peptide bonds (c) A protein is exposed to extremely high heat, causing it to lose its secondary structure and be left with only its primary structure. (d) Amino acids fold into repeating patterns due to hydrogen bonding of the peptide backbone. 2. Protein A will fold into its native state only when protein B is also present in the solution. However, protein B can fold itself into native confirmation without the presence of protein A. Which of the following is true? (a) Protein B serves as precursor for protein A (b) Protein B serves as molecular chaperon for protein A (c) Protein B serves as ligand for protein A (d) Protein B serves as structural motif for protein A 3. Unfolding of a protein can be termed as _________ (a) Denaturation (b) Renaturation (c) Reduction (d) Oxidation  Haemoglobin Structure and function of Myoglobin http://upload.wikimedia.org/wikipedia/commons/thumb/6/60/Myoglobin.png/250px-Myoglobin.png Haemoglobin and Myoglobin Fall 2024-2025  Haemoglobin Haemoglobin is the iron-containing protein attached to red blood cells that transports oxygen from the lungs to the rest of the body. Haemoglobin is an example of both globular and quarternary structure of protein.  Haemoglobin 1. Haemoglobin has 4 polypeptide chains. 2. Two of these chains, called α-polypeptides are identical and each consists of 141 amino acids. 3. The other two chains, called β-polypeptides, also identical and each consists of 146 amino acids  Haemoglobin 4. Each polypeptide has a haem group, which contains iron that binds to oxygen. 5. Each haemoglobin can carry 4 molecules of oxygen. 6. When haemoglobin combines with oxygen it forms a molecule called oxyhaemoglobin 7. Hydrophobic interactions of groups within the haemoglobin molecule maintain haemoglobin shape  Myoglobin http://upload.wikimedia.org/wikipedia/commons/thumb/6/60/Myoglobin.png/250px-Myoglobin.png Myoglobin is a single-chain - - Globular protein of 153 amino acids -- It contains a haem (iron-containing group) prosthetic group in the center around which the remaining apoprotein folds. It has eight ----- alpha helices and a hydrophobic core. It is the primary oxygen-carrying pigment of muscle tissues Myoglobin Myoglobin has a stronger affinity for oxygen than haemoglobin. The presence of myoglobin gives meat its bright red color. The heme is held in position by the bonding of a nitrogen on a histidine side chain from the protein to iron in heme. A second histidine is in the vicinity on the opposite side of the heme but is not bonded. There is one bonding position on iron ion for the attachment of oxygen diatomic molecule  Practice 9 Compare Haemoglobin & Myoglobin Haemoglobin Myoglobin  Protein digestion 1. Mouth : crushed and moistened by saliva 2. Stomach : Proteins are denatured by hydrochloric acid. Pepsinogen is activated to pepsin and pepsin begins the digestion of proteins into aminoacids, dipeptides and tripeptides (10- 20% of digestion). 3. Small intestine Pancreatic and intestinal proteases and peptidases complete digestion. The cells of the small intestine produce peptidases to complete digestion, absorb amino acids, and release them into the bloodstream for use by the body’s cells.  Practice 10 Match the following 1. Proteoses and peptones A) amide group containing amino acid - L 2. Albumin B) Present k - in stomach 3. Nucleoprotein L C) Derived proteins # 4. Primary structure of protein G D) Zwitterion # 5. Alpha helix J E) Molecular Chaperons - 6. Tertiary structure of protein H F)- Basic amino acid 7. A molecule which carries positive and G) Keratin & negative charges at the same time in equal D number 8. Heat shock proteins E H) Enzyme Lipase 9. Diaminomonocarboxylic acid f I) Silk fibroin - 10. β - pleated sheet I J) Insulin 11. Asparagine A K) Simple protein example - 12. Pepsin B L) Conjugated protein - Thank you Any questions?  CHAPTER-2 Protein Purification BIOCHEMISTRY BIOC 210 Biochemistry BIOC210  Why purify a protein? To study its function To analyze its physical properties To determine its sequence For industrial or therapeutic applications. How can proteins be extracted from cells? Many steps/techniques are needed to extract and separate protein of interest from many contaminants. Before purification begins, protein must be released from cell by homogenization. Biochemistry BIOC210  What are different ways of homogenization of cells? Grinding tissue in a blender with a suitable buffer - Releases soluble proteins and various subcellular organelles B Potter-Elvejhem homogenizer – A thick-walled test tube with a tight-fitting plunger - Breaks open cells – organelles intact Sonication – Sound waves to break open cells Continuous freezing and thawing – Ruptures cells The choice of which way is best depends upon the cell type to be processed (E.coli, yeast, heart/skin tissue, RBCs). Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 4 BIOC210 4  Once proteins are extracted out from their cellular environments They are unstable: May get denatured (loss of structure and activity) May get proteolysed by several proteases released from cell rupture May get modified (oxidized) To avoid this most of the time, all purification steps are carried out: At 0-4 oC and in presence of general protease inhibitors. Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 5 BIOC210  Differential Centrifugation Differential centrifugation is a method used to separate the different components of a cell on the basis of mass. The homogenate is centrifuged to obtain a pellet containing the most dense organelles. Compounds that are the most dense will form a pellet at lower centrifuge speeds while the less dense compounds will likely remain in the liquid supernatant above the pellet Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 6 BIOC210 6  Each time, the supernatant may be centrifuged at faster speeds to obtain the less dense organelles. Performing centrifugation in a stepwise fashion, in which the centrifugation speed is increased each time, allows the components to be separated by mass. The rather dense nucleus is most likely to be found after the first centrifugation step, followed by the mitochondria, then smaller organelles, and finally the cytoplasm, which may contain soluble proteins. Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 7 BIOC210 7  Differential Centrifugation Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida BIOC210  Differential Centrifugation Name the homogenizer shown above ?? Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 9 BIOC210  Zonal centrifugation (density gradient) Use of continuous density gradient of solvent such as sucrose. Density increases towards the bottom of the tube. Sample layered on the top. Molecules form discrete bands after centrifugation. Separation based on size of the molecules. Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 10 BIOC210 10  Purification Techniques All purification techniques utilize different properties of proteins like: S 1. Solubility. -Salting in and Salting out 2. Charge (ionic) -Ion exchange chromatography - Electrophoresis - Isoelectric focusing electrophoresis 3. Molecular size -Size exclusion gel filtration 4. Binding specificity -Affinity chromatography Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 11 BIOC210 11  Separation based on solubility Salting In and Out The solubility is also a function of ionic strength Salting-in: Salting-out: At low salt concentrations, the As you increase the ionic presence of salt stabilizes strength by adding salt, there various charged groups on a is less and less water available protein molecule, thus for the protein to dissolve and attracting protein into the proteins will precipitate solution and enhancing the solubility of protein. Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida BIOC210 12  Ammonium sulfate (NH4SO4) commonly used to “salt out” Takes away water by interacting with it, makes protein less soluble because hydrophobic interactions among proteins increases and they precipitate out. Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 13 BIOC210 13  Column Chromatography Important steps in column chromatography 1.Pack column - Column is packed with material (resin) that can absorb molecules based on some property (charge, size, binding affinity, etc.) 2.Wash column - Molecules washed through the column with buffer 3.Collect fractions - Fractions are taken, at some point your molecule will elute Note : This technique is used in ion exchange chromatography (based on charge), Size exclusion gel filtration chromatography (based on size) and Affinity chromatography (based on specific binding). Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 14 BIOC210 14  All the methods shown below use Column Chromatography Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida BIOC210 15  Column Chromatography Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 16 BIOC210 16  Ion exchange chromatography (Separation based on charge) Ion exchange resins contain charged groups. 1. Cation exchangers They are acidic in nature They are negatively charged They interact with positively charged proteins 2. Anion exchangers They are basic in nature They are positively charged They interact with negatively charged proteins Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida BIOC210 17  Ion exchange chromatography For protein binding, the pH is fixed (usually near neutral) under low salt conditions. Example cation exchange column… + Positively charged protein or - CH2-COO + enzyme bind to the column CH2-COO- + + CM cellulose cation exchanger - - - - Negatively charged - proteins pass - through the - column - Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 18 BIOC210 18  Ion exchange chromatography To elute our protein of interest, add increasingly higher amount of salt NaCl (increase the ionic strength). Na+ will interact with the cation resin and Cl- will interact with our positively charged protein to elute off the column. CH2-COO- + + Increasing [NaCl] + CH2-COO- of the elution + buffer + CM cellulose cation exchanger CH2-COO- Na+ Na+2 Cl- Cl- + CH2-COO- Na+ - + Cl + CM cellulose Na+2 Cl- + Biochemistry cation exchanger BIOC210 Prepared By Mrs. Anjum Khatoon & Dr. Nida 19 19  Ion exchange chromatography using stepwise elution Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 20 BIOC210 20  Practice-1 Which protein will be collected in the beaker B in the first elution ? Name the type of exchanger used in the column ? Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida BIOC210 21  Electrophoresis (separation based on charge) Electrophoresis: charged particles like proteins and nucleic acids migrate in an electric field towards the opposite charge. Proteins have different mobility depending on: – Charge – Size – Shape Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 22 BIOC210 22  Electrophoresis Electrophoresis is usually done with gels formed in tubes, slabs, or on a flat bed. In many electrophoresis units, the gel is mounted between two buffer chambers containing separate electrodes, so that the only electrical connection between the two chambers is through the gel. Because the pores of an agarose gel are large, agarose is used to separate macromolecules such as nucleic acids, large proteins and protein complexes. Polyacrylamide, which makes a small pore gel, is used to separate most proteins and small oligonucleotides. Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 23 BIOC210 23  Practice -2 Looking at Picture (B) predict and draw the movement of molecules in (C) Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida BIOC210 24  Isoelectric Focusing (Separation based on charge) Isoelectric Point: There is a pH at which there is no net charge on a protein, this is the isoelectric point (pI).(chapter-1) Isolectric focusing- based on differing isoelectric pts (pI) of proteins. Gel is prepared with pH gradient that parallels electric-field. Charge on the protein changes as it migrates. When it gets to pI, and has no charge the protein stops migrating. Separated and identified on differing isoelectric pts. (pI) Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 25 BIOC210 25 Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 26 BIOC210 26 Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida BIOC210  Practice -3 In figure (A) different proteins with charges are shown. Mark in figure B the the position of the proteins that have attained isoelectric points (pI) O o or Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida BIOC210 28  The pI of some proteins are shown in the table. Mark the position of the proteins - Pepsin, Urease, Myoglobin, Lysozyme in the picture (P) Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida BIOC210 29  Size-Exclusion/Gel-Filtration Separates molecules based on size. Stationary phase composed of cross-linked gel particles.(beads) Extent of cross-linking can be controlled to determine pore size Smaller molecules enter the pores and are delayed in elution time. Larger molecules do not enter and elute from column before smaller ones. removed Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 30 BIOC210 30 Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 31 BIOC210 31  Affinity Chromatography Based on binding affinity (high selectivity of most protein). Uses specific binding properties of molecules/proteins. Stationary phase has a polymer that can be covalently linked to a compound called a ligand that specifically binds to protein. Ligands bind to desired protein or vice versa. Proteins that do not bind to ligand elute out. Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 32 BIOC210 32 Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 33 BIOC210 33 Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida BIOC210  Applications of purified proteins Purified proteins are used therapeutically to treat patients suffering from various diseases Like: cancers, heart attacks, strokes, cystic fibrosis, diabetes, anemia, hemophilia, etc. The first protein used to treat disease was insulin. Theraputic proteins Potential applications Insulin Diabetes mellitus Human growth Treating factor hypopituitary dwarfism in children Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 35 BIOC210 35  Practice-4 (Matching) 1. Affinity Chromatography A) Uses Sound waves I 2. Electrophoresis B) Separation based on mass i 3. Differential Centrifugation C) Uses temperature changes 4. Salting in and Salting out D) Uses Cation/anion exchangers & 5. Isoelectric focusing E) Separation based on size 6. Ion-exchange chromatography F) Breaking open/lysis of the cells 7. Zonal Centrifugation G) Uses pH gradients 8. Size exclusion Chromatogrphy H) Uses Agarose gel 9. Cell Homogenization F I) Uses Ligands/ highest affinity 10. Sonication A J) Separation based on solubility 11. Potter-Elvejhem homogenizer L K) Separation based on density 12. Freezing and thawing [ L) & Uses thick-walled test tube & Plunger Biochemistry Prepared By Mrs. Anjum Khatoon & Dr. Nida 36 BIOC210 36  MOHAMMED AL-MANA COLLEGE FOR MEDICAL SCIENCES BIOC 210 1 Chapter - 3 Enzymes 1  Enzymes Enzymes are biological catalysts responsible for supporting almost all of the chemical reactions that maintain body homeostasis. Enzymes are globular proteins. Enzymes are proteins and their activities depends on the 3D structure of the amino acids that compose them. 3  The enzyme active site One part of an enzyme, the active site, is particularly important It is a pocket in the enzyme where substrates binds via noncovalent interactions, and catalytic reaction occurs. The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily The active site can discriminate among possible substrate molecules. 4  The substrate The substrate of an enzyme are the reactants that are activated by the enzyme Enzymes are specific to their substrates The specificity is determined by the active site 5 Substrate Binding Types of Reactions Single Substrate - Single Product : A ⇄ B Single Substrate - Multiple Products : A ⇄ B + C Multiple Substrates - Single Products : A + B ⇄ C Multiple Substrates - Multiple Products : A + B ⇄ C + D Ordered Random Multiple Substrate Binding Ordered Random NADH + Pyruvate Creatine + ATP No order to binding Creatine phosphate + ADP Must Lactate + NAD+ 6 bind Lactate Creatine first Dehydrogenase Kinase  Practice- 1 Which of the following best describes The active site of an enzyme? a) Is an organic molecule on the enzyme b) is complementary to the rest of the molecule. c) contains amino acids without sidechains. d) Is a specific shape on the enzyme 7  Properties of Enzymes All Enzymes are Proteins Enzymes are biological catalysts They lose their activity if denatured They lower the activation energy of the reaction The small quantity of enzymes catalyzes the larger quantities of substances. Enzymes increase the rate of reaction and remain unaffected by the reaction which they catalyze May contain cofactors such as metal ion or organic (vitamins) The enzyme molecule can be reused repeatedly as they are not consumed by the reaction Enzymes are highly specific in nature, (a particular enzyme can catalyze a particular reaction) 8 Match the following words with their definitions Product [ 4 ] 1. Amount of energy required for a chemical reaction to occur. Active site [ 3 ] 2. Substance that enzymes act upon Enzymes [ 3 ] 3. Regions on the surface of enzymes that fit the substrate. Substrate [ 2 ] 4. Substance formed from the Activation Energy [ I ] substrate at the end of a chemical reaction with an enzyme. 5. Proteins that speed up chemical reactions. ↑ 10  Label the diagram with these terms : Enzyme, Product, Substrate, Active site 11  Enzyme Cofactors Some enzymes depend for activity only on their structure as proteins while others require one or more nonprotein components called cofactors. The protein part of the enzyme is called apoenzyme The non-protein part cofactor together with the protein part apoenzyme forms a holoenzyme Apoenzyme + Cofactor → Holoenzyme (protein part) (non-protein) 12  The cofactors can be divided into 2 types 1. a metal ion E.g., Zn2+, Cu2+, Mn2+, K+, Na + 2. an organic molecule called a coenzyme. E.g., The B group vitamins like B1, B2 , NADH, CoA ….. 13  Practice -2 1. The enzyme carboxypeptidase is synthesized in the pancreas and secreted into the small intestine. This enzyme hydrolyzes the first peptide bond at the C-terminal end of proteins and peptides. The enzyme has zinc ion tightly bound to the active site. The zinc ion is called a) Product b) Cofactor c) Coenzyme d) Substrate 14  Practice-3 Guess the name of the enzymes from the list to their substrates and functions 1. Digest milk (lactose) ……………….. 2. Digest starch (amylose) ………………. 3. Synthesizes ATP …………….. 4. Digests Protein ……………… ② D Amylase, Protease, Lactase, DNA polymerase, ATP Synthase, Maltase B 16  Classification of Enzymes Class Reactions catalyzed Oxidoreducta oxidation-reduction/ add or remove hydrogen atoms. ses A + B: ↔ A: + B Pyruvate + NADH → Lactic acid + NAD+ Lactate dehydrogenase Transferases Transfer groups between donor and acceptor molecules A + BX → AX + B Met-Ala-Leu + Lys-tRNA → Met-Ala-Leu-Lys + t RNA Peptidyl transferase Hydrolases Add water across a bond, hydrolyzing it. A + H2O → B + C Lys-Ala + H2O → Lys + Ala Serine proteases 17  Classification of Enzymes Class Reactions catalyzed Lyases Catalyzes the breaking of various chemical bonds by means other than hydrolysis and oxidation. A→B+C Arginosuccinate → Arginine + Succinate Arginosuccinate lyase Isomerases Carry out many kinds of isomerization: L to D isomerization, mutase reactions (shifts of chemical groups) and others. A→B Dihydroxyacetone phosphate → Glyceraldehyde-3-phosphate Triosephosphateisomerase Ligases Catalyze reactions in which two chemical groups are joined (or ligated) with the use of energy from ATP. A + B → A-B Acetate + CoA +ATP → Acetyl CoA+AMP Acetyl CoA Synthetase 18 Group activity-2 19  Group activity-2 A study was done to investigate for the mechanism of the enzymes as shown in the following 3 reactions. What would you classify each enzyme? (1) isomerase hydrolase (3) oxidoreductase 20  How enzymes work ? Enzymes are biological catalysts. -Enzymes enhance the reaction rates (molecules produced per second) by lowering the activation energy of the transition state How does an enzyme lower Ea? By Stabilizing the Transition State! Puts molecule in close proximity to react Puts molecule in correct orientation 21 First Step: Enzyme binds to substrate molecule to form an enzyme–substrate complex E + S ↔ES Second Step: Formation of the transition state complex where the bound substance is neither product nor reactant ES↔ES* Third Step: Formation of the enzyme –product complex ES* → EP Fourth Step: Release of product EP → E + P 22  TWO MODELS FOR ENZYME/SUBSTRATE INTERACTIONS: The mechanism of enzyme action is explained by two hypothesis 1. Lock and Key model 2. Induced Fit model 23  Enzyme Action Lock and Key Model An enzyme-substrate complex forms when the enzyme’s active site binds with the substrate like a key fitting a lock. The shape of the active site of the enzyme must be complementary to the shape of the substrate. Enzymes are therefore very specific; they will only function correctly if the shape of the substrate matches the active site. 24  Lock and Key Model This explains the loss of activity when enzymes denature. Products have a different shape from the substrate Once formed, they are released from the active site Leaving it free to become attached to another substrate 25  Enzyme Action:Induced Fit Model Some proteins can change their shape (conformation) When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation The active site is then moulded into a precise conformation Making the chemical environment suitable for the reaction The bonds of the substrate are stretched to make the reaction easier (lowers activation energy) This explains the enzymes that can react with a range of substrates of similar types 26  Name the Enzyme Model A and B A B 27 Differentiate between Lock & Key and Induced Fit Model Lock and Key Induced Fit Model 28  Factors Affecting Enzyme Action Factors that influence reaction rates of Enzyme catalyzed reactions include 1. Enzyme and substrate concentrations 2. Temperature 3. pH 29 Effect of Substrate Concentration At low concentrations, an increase in substrate concentration increases the rate because there are many active sites available to be occupied At high substrate concentrations the reaction rate levels off because most of the active sites are occupied 30 Effect of Temperature Enzymes are thermolabile or heat-sensitive. Rate increases with temperature Above a certain temperature, the rate begins to decline because the enzyme protein begins to denature Most active at optimum temperatures (usually 37°C in humans) 31  Effect of pH Each enzyme has an optimal pH at which it is most efficient. A change in pH can alter the ionization of the R groups of the amino acids. When the charges on the amino acids change, hydrogen bonding bonding within the molecule will change and the structure of the enzyme is changed. Pepsin is most efficient at pH 2 – 2.5 The active site is distorted and while Trypsin is much higher pH 8 – 8.5 the substrate molecules will no longer fit in it. Extreme pH levels will produce denaturation 32  Practice - 5 1. From the data below What is the optimum pH for the pyruvate dehydrogenase enzyme? Optimum pH Activity % Relative Activity pH 33 2. You are studying the effects of temperature on the rate of enzyme catalyzed reaction. When you decrease the temperature from 35°C to 28 °C, what effect will this have on the rate of reaction ? a) It will increase b) It will decrease c) It will decrease to zero because the enzyme denatures d) It will not change 3. If an enzyme solution is saturated with substrate, the most effective way to obtain an even faster yield of product is to a) Add more of the enzyme b) Heat the solution c) Add more of substrate d) Add allosteric inhibitor 34  Applications of enzymes Since enzymes activity is essential for living cells, any malfunction can cause genetic diseases (phenylketonuria,….etc). 1. Oral adminstration of enzymes can be used to treat several diseases. Ex: Pancreatic enzymes may be given to treat digestive problems resulting from removal of the pancreas or certain diseases of the pancreas. 2. Contact lens cleaners (proteases), to remove proteins on contact lens to prevent infections. 35  Applications of enzymes…….. 3. Biological detergent (lipases), used to assist in the removal of fatty and oily stains. 36  Enzyme Inhibition Enzyme inhibitors block enzyme action. A chemical that interferes with enzyme’s activity is called an inhibitor. Irreversible inhibition – an inhibitor attaches to the enzyme by covalent bonds. Reversible inhibition – an inhibitor attaches to the enzyme by weak bonds. There are 2 types of enzyme inhibitors. Competitive inhibitor Noncompetitive inhibitor 37  Competitive Inhibition revisable A competitive inhibitor Has a structure similar to substrate Occupies active site Competes with substrate for active site Has effect reversed by increasing substrate concentration 38  Noncompetitive Inhibition A noncompetitive inhibitor Does not have a structure like substrate Binds to the enzyme but not active site. Changes the shape of enzyme and active site Substrate cannot fit altered active site No reaction occurs Effect is not reversed by adding substrate 39 40  Practice - 6 Differentiate between Competitive & Non-competitive Inhibitor Competitve Inhibitor Non-Competitive Inhibitor 41 Practice ……. 42  Feed Back Inhibition Inhibitors are not always harmful. Enzyme inhibition is an important mechanism for regulating cell metabolism. A metabolic pathway involves a series of steps catalyzed by enzymes to form a product. The over-produced product may act as an inhibitor of one of the enzymes of the pathway. The inhibition, whereby a metabolic reaction is 43 blocked by its products, is called feedback inhibition. 43 Feedback inhibition : The inhibition, whereby a metabolic reaction is blocked by its products, is called feedback inhibition. 44  Allosteric enzymes Allosteric enzymes change shape between active and inactive shapes as a result of the binding of substrates at the active site, and of regulatory molecules at other sites. Activators and inhibitors are termed "effectors“ or “modulators”. Inhibitors cause the allosteric enzyme to adopt the inactive shape. Activators promote the active shape. 45  Allosteric Regulation Allosteric regulation controls an enzyme’s activity. Allosteric regulation is the term used to describe cases where a protein’s function at one site is affected by binding of a regulatory molecule or modulator at another site. Allosteric regulation may either inhibit or stimulate an enzyme’s activity by changing an enzyme into its active or inactive forms. 46  Applications of inhibitors Negative feedback: end point or end product inhibition Poisons snake bite, plant alkaloids and nerve gases. Medicine antibiotics, sulphonamides, sedatives and stimulants 47  Practice-7 Activity of allosteric enzymes are influenced by a. Allosteric modulators b. Allosteric site c. Catalytic site d. None of the above Feed back inhibition means. e. Initial product inhibition f. End Product inhibition g. Enzymatic induction h. None of the above 48  Ahmed was studying the effect of the temperature of a specific enzyme as shown below. He expressed the enzyme activity in the pie graph, Use the data to predict what is the optimum temperature of this enzyme? Under Which class you may classify this enzyme? Green: 30 , 29, 37, 44 49