Biochemistry Basic Molecules and enzymes 2024-25 PDF

Biochemistry (Basic molecules and enzymes) Syllabus Section 2 Dr Michelle Ellul Biomolecules: Basic molecules and enzymes Dr M. Ellul Basic Chemistry Learning outcomes Define an element Def...

Biochemistry (Basic molecules and enzymes) Syllabus Section 2 Dr Michelle Ellul Biomolecules: Basic molecules and enzymes Dr M. Ellul Basic Chemistry Learning outcomes Define an element Define and describe atoms Explain where electrons are found in an atom Define isotopes Matter refers to anything that takes up space and has mass. All matter, both living and nonliving, is composed of certain basis substances called elements. Elements are substances that cannot be broken down into substances with different properties (chemical or physical). The most common elements found in living organisms are: carbon, hydrogen, oxygen, nitrogen, phosphorus and sulfur. Elements contain atoms. There is only one type of atom in each element. Atoms are the smallest units of a substance. They are composed from a central atomic nucleus and electrons that orbit around the nucleus in electron shells. Protons Neutrons Electrons Charge Positive (+1) No charge (neutral / 0) (Negative (-1) Location Nucleus Nucleus Orbits / electron shells Weight One atomic mass A little more than one Negligible mass unit atomic mass unit Overall, the atom does not have any charge as the number of protons and the number of electrons in an atom is equal. The atomic number (or proton number) is the number of protons in the nucleus of an atom and has symbol Z The atomic number is equal to the number of electrons present in a neutral atom of an element. 2 Biomolecules: Basic molecules and enzymes Dr M. Ellul The mass number is the total number of protons and neutrons in the nucleus of an atom and has symbol A Isotopes are atoms of the same element that contain the same number of protons and electrons but a different number of neutrons. Electrons (negatively charged) move around the nucleus in regions called electron shells or energy levels. Shells are filled in order: Electrons always occupy the lowest available energy level (shell) first. The shells are filled starting from the one closest to the nucleus, moving outward. First shell (closest to the nucleus) can hold up to 2 electrons. Second shell can hold up to 8 electrons. Third shell can also hold up to 8 electrons (though, in chemistry, it can hold more under specific conditions). Electron shells determine how atoms interact with each other. Atoms with incomplete outer shells (called valence shells) are more likely to react and form chemical bonds. Chemical bonds are crucial in biology for forming molecules like DNA, proteins, carbohydrates, and fats, which are the building blocks of life. 3 Biomolecules: Basic molecules and enzymes Dr M. Ellul When atoms of two or more different elements react or bond together, a compound results. Chemical bonds Learning outcomes Describe ionic bonding Explain how covalent bonds form molecules Contrast polar and nonpolar covalent bonds Describe hydrogen bonding Chemical bonds form when there are forces of attraction between atoms. Ionic bonding When metals react with non-metals, electrons are transferred from the metal atoms to the non- metal atoms. As a result, there is the formation of ions. E.g. Sodium + chlorine → sodium chloride Metal Non-metal salt 4 Biomolecules: Basic molecules and enzymes Dr M. Ellul Sodium Chlorine became a became a positively negatively charged charged particle particle Ionic bonding (Adapted from: https://www.bbc.co.uk/bitesize/guides/z6k6pbk/revision/1) An ion is a charged particle produced by transfer of electrons from a metal atom to a non-metal atom. There is a strong electrostatic force of attraction between these oppositely charged ions – this is called an ionic bond. Positively charged ions are called cations; negatively charged ions are called anions. Covalent bonding A covalent bond forms when two non-metal atoms share a pair of electrons. The electrons involved are in the outer shells of the atoms. An atom that shares one or more of its electrons will complete its outer shell. 5 Biomolecules: Basic molecules and enzymes Dr M. Ellul Covalent bonding (Adapted from: https://www.bbc.co.uk/bitesize/guides/z6k6pbk/revision/2) A molecule consists of two or more atoms chemically bonded together by covalent bonding. Polarity and hydrogen bonding Normally the sharing of electrons between two atoms is equal and the covalent bond is nonpolar. In some cases, e.g. water, the sharing of electrons between oxygen and each hydrogen is not completely equal. The larger oxygen atom has a greater number of protons and so attracts more electrons towards itself. The attraction of an atom for the electrons of a covalent bond is called electronegativity. The oxygen atom is more electronegative than the hydrogen atom, and it can attract the electron pair to a greater extent. In the water molecule, this causes the oxygen atom to assume a slightly negative charge (δ-) and it causes the hydrogen atom to assume a slightly positive charge (δ+). The unequal sharing of electrons in a covalent bond creates a polar covalent bond. Polarity within a water molecule causes the hydrogen atoms in one molecule to be attracted to the oxygen atoms in other molecules. This attractive force creates a weak bond called a hydrogen bond. 6 Biomolecules: Basic molecules and enzymes Dr M. Ellul Biological molecules can contain many polar covalent bonds involving hydrogen and usually oxygen or nitrogen. Van der Waals forces Nonpolar molecules interact very little with polar molecules, including water. Nonpolar molecules are attracted to one another by very weak bonds called van der Waals forces. 7 Biomolecules: Basic molecules and enzymes Dr M. Ellul 2.1.1: Water Syllabus: The dipole nature; an awareness that the collective effect of the hydogen bonds is responsible for the unique properties of water exemplified by importance of water as a solvent and its biological significance. A very brief mention of the other biologically significant properties of water. 8 Biomolecules: Basic molecules and enzymes Dr M. Ellul Learning outcomes Describe the molecular structure of water. Discuss polarity in water. Show how hydrogen bonding affects the properties of water. Discuss the relevance of water’s unusual properties for living systems. Without water, life could not exist on this planet. It is important to living organisms because it is both a vital chemical constituent and for many, a habitat. Molecular structure of water: Water consists of an oxygen atom bound to two hydrogen atoms by two single covalent bonds. The electronegativity of O is much greater than that of H and the bonds between these atoms are highly polar. Hydrogen bonds form between the partially negative O atoms and the partially positive H atoms of two water molecules. Hydrogen bonds are responsible for unique properties of water. Shape of a water molecule: The two covalent bonds of a water molecule have a partial charge at each end. The most stable arrangement of these charges is a tetrahedron. The H-O-H angle is 104.5, which is slightly less than the bond angle of a perfect tetrahedron. This is due to the fact that the partial negative charges occupy more space than the partial positive regions. 9 Biomolecules: Basic molecules and enzymes Dr M. Ellul Water as a solvent: Water is an extremely good solvent. It can dissolve a wide range of substances, especially polar molecules and ionic compounds. Water is a polar molecule, and it has positive and negative ends. If a salt (NaCl) crystal is dropped into water, the positively charged hydrogen ends of water molecules will be attracted to and surround chloride ions, and the negatively charged oxygen ends of water molecules will surround the positively charged sodium ions. The slightly charged regions of the water molecule surround atoms of opposing charge, forming dispersive hydration shells. Water dissolves polar molecules as its positive and negative poles are attracted to oppositely charged regions of dissolving molecules. Charged and polar molecules are termed hydrophilic. Many biological molecules, such as sugars and amino acids are hydrophilic and dissolve readily in water. E.g. glucose is composed of molecules that contain polar hydroxyl (OH) groups. Water molecules can form hydrogen bonds with individual hydroxyl groups of the glucose molecules. Water molecules surround it forming a hydration shell. Water also dissolves gases such as oxygen and carbon dioxide. Why is solubility of water important to life? _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________ 10 Biomolecules: Basic molecules and enzymes Dr M. Ellul Molecules that are uncharged and non-polar, such as fats and oils, usually do not dissolve in water and are called hydrophobic. Nonpolar molecules tend to aggregate (clump together) in water, and this is known as hydrophobic exclusion. This causes these nonpolar molecules to form certain shapes. Non-polar substances such as lipids are immiscible with water and can serve to separate aqueous solutions into compartments, as with membranes. Hydrophobic exclusion also affects the structure of DNA and proteins. Reflect on what you learnt: Discuss why the interaction of water with both nonpolar and polar molecules (and ions) is critical to living systems. Other biologically significant properties of water High heat capacity: The specific heat capacity of water is the amount of heat, measured in joules, required to raise the temperature of 1 kg of water by 1oC. Water has a high heat capacity. This means that a large increase in heat energy results in a relatively small rise in temperature. This is because much of the energy is used in breaking the hydrogen bonds that restrict the mobility of the molecules. Temperature changes within water are minimized because of its high heat capacity. Why is this important for living organisms? 11 Biomolecules: Basic molecules and enzymes Dr M. Ellul High latent heat of vaporisation: A relatively large amount of energy is needed to vaporise water. A cooling effect occurs. This is made use of in sweating and panting of mammals. The high heat of vaporisation means that a large amount of heat can be lost with minimal loss of water from the body. High latent heat of fusion: Latent heat of fusion is a measure of the heat energy required to melt a solid, in this case ice. Water requires relatively large amounts of heat energy to thaw it. Conversely, liquid water must lose a relatively large amount of heat energy to freeze. Contents of cells and their environments are therefore less likely to freeze. Density and freezing properties: Ice is less dense than liquid water, so when a pond or lake starts to freeze in water, ice stays at the top, forming an insulating layer that delays the freezing of the rest of the water. If ice sank, ponds and lakes of cold countries would freeze during the winter, eliminating fish and making liquid drinking water far less available to animals. High surface tension and cohesion: Water has high cohesion – that is, water molecules tend to stick together. (Water molecules sticking to other types of molecules is called adhesion). Cohesion among water molecules at the surface of ponds and lakes produces surface tension. The high cohesion of water molecules is important in cells and in translocation of water through xylem in plants. Many small organisms rely on surface tension to settle on water or to shake over its surface. Water as a reagent: Water enters into many of the chemical reactions that occur in living cells. Water is used as a source of hydrogen in photosynthesis. 12 Biomolecules: Basic molecules and enzymes Dr M. Ellul Some biologically important functions of water All organisms Structure - high water content of protoplasm Solvent and medium for diffusion Reagent in hydrolysis Support for aquatic organisms Fertilisation by swimming gametes Dispersal of seeds, gametes and larval stages of aquatic organisms and seeds of some terrestrial species Plants Osmosis and turgidity for support, guard cell mechanism Reagent in photosynthesis Transpiration and translocation of inorganic ions and organic compounds Germination of seeds – swelling and breaking open of the testa and further development Animals Transport Osmoregulation Cooling by evaporation, such as sweating, panting Lubrication, as in joints Support – hydrostatic skeleton Protection, for example lachrymal fluid, mucus Migration in ocean currents Self-assessment 1. Draw a few water molecules. Label the types of bonds found in and between the molecules. 2. How does electronegativity affect the interactions between water molecules? 3. Describe the properties of water, give an example of each. 4. Describe two ways in which the properties of water benefit organisms. 5. Imagine if O and H had the same electronegativity, what would that do to the properties of water? 13 Biomolecules: Basic molecules and enzymes Dr M. Ellul Basic Chemistry of Carbon Learning outcomes Identify the types of bonds found in organic compounds. Identify the major functional groups found in biomolecules. Define the term isomer. Distinguish between the different types of isomers. Distinguish between monomers and polymers. Distinguish between condensation and hydrolysis reactions. The most common elements in living organisms are carbon, hydrogen, nitrogen and oxygen. Organic molecules are those molecules that contain carbon and hydrogen bonds. Carbon has 4 electrons in its outer shell and it can bond with as many as four other atoms. Usually carbon bonds to hydrogen, oxygen, nitrogen or to another carbon atom. These bonds are covalent. The ability of Carbon to bind to itself makes carbon chains of different lengths and shapes possible. Carbon can also share two pairs of electrons with another atom forming a double bond. Triple bonds can also form. Functional groups Hydrocarbon chains make up the backbone (skeleton) of the organic molecules. Functional groups can be attached to the carbon chain. Functional groups are clusters of certain atoms that always behave in a certain way. 14 Biomolecules: Basic molecules and enzymes Dr M. Ellul Hydrocarbon chains (composed only of carbon and hydrogen) are hydrophobic. A functional group that can form ions can make an organic molecule hydrophilic. Isomers Isomers are molecules that have identical molecular formula, but they are different molecules because the atoms in each are arranged differently. There are different types of isomers: Structural isomers differ in the placement of their covalent bonds - the different arrangement of the atoms within the molecules leads to differences in their chemical properties. Geometric isomers or stereoisomers, on the other hand, have similar placements of their covalent bonds but differ in how these bonds are made to the surrounding atoms, especially in carbon-to-carbon double bonds. Structural isomers Stereoisomers Certain solid compounds when dissolved to form a solution possess the power to rotate the plane of vibration of plane-polarised light, and are said to be optically active. 15 Biomolecules: Basic molecules and enzymes Dr M. Ellul If the substance rotates the plane of polarisation to the right, it is said to be dextro-rotatory (D- isomer) and if to the left laevo-rotatory (L-isomer). Optical isomerism is a property of any compound which can exist in two forms whose structures are mirror images. In organic compounds, this occurs when a carbon atom has four different atoms or groups attached to it. Such a carbon atom is called an asymmetric carbon atom. A compound which has mirror images is called a chiral compound. e.g. lactic acid The two mirrored forms are known as stereoisomers, true stereoisomers cannot be superimposed on each other, two forms are known and D- and L- forms. In general sugars within living organisms are of the D form, amino acids are of the L form. Monomers and polymers Monomers are the smaller units from which larger molecules are made. Polymers are molecules made from a large number of monomers joined together in a chain. Carbon compounds can form small single subunits (monomers) that bond with many repeating subunits to form large molecules (polymers) by a process called polymerisation. Macromolecules are very large molecules. They contain 1000 or more atoms and so have a high molecular mass. Polymers can be macromolecules, however, not all macromolecules are polymers as the subunits of polymers have to be the same repeating units. Formation of polymers A condensation reaction occurs when monomers combine together by covalent bonds to form polymers (polymerisation) or macromolecules and water is removed. Condensation is also known as dehydration synthesis. 16 Biomolecules: Basic molecules and enzymes Dr M. Ellul Breaking down of polymers Polymers are broken down by a reaction known as hydrolysis. Hydrolysis means ‘lyse’ (to break) and ‘hydro’ (with water). In the hydrolysis of polymers, covalent bonds are broken when water is added. Self-assessment 1. What are isomers? 2. List and describe the different types of isomers. 3. Distinguish between monomer and polymer. 4. Differentiate between a hydrolysis and a condensation reaction. 17 Biomolecules: Basic molecules and enzymes Dr M. Ellul 2.1.2: Carbohydrates Syllabus: Monosacharides: pentoses (ribose, deoxyribose) detail of structures not examinable; hexoses (glucose, fructose and galactose), basic distinction between the structures of α-glucose and β – glucose. Disaccharides: (maltose, sucrose, lactose); 1,4 glycosidic linkages exemplified by maltose. Polysaccharides: Basic structure of starch, cellulose and glycogen related to function. 18 Biomolecules: Basic molecules and enzymes Dr M. Ellul Learning outcomes Describe the structure of monosaccharides. Distinguish between α-glucose and β –glucose. Describe how 1,4 glycosidic linkages result in the formation of disaccharides. Describe the basic structure of starch, cellulose and glycogen and relate it to function. Carbohydrates are substances with the general formula C x(H2O)y, where x and y are variable numbers. All carbohydrates are aldehydes or ketones. All contain several OH groups. Aldehydes are easily oxidised and therefore they are powerful reducing agents. Monosaccharides Monosaccharides are single sugar units with a carbon backbone of three to seven carbon atoms. Glyceraldehyde is a carbohydrate that contains 3 carbon atoms. It is a triose. Most common monosaccharides are pentoses (e.g. ribose and deoxyribose) and hexoses (e.g. glucose, fructose, and galactose). Pentoses have 5 carbons while hexoses have 6 Carbon atoms. Ribose and deoxyribose have 5 carbons and are part of the genetic molecules ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Glucose is the most common monosaccharide in living organisms and is the subunit of which most polysaccharides are made. Glucose has 6 carbons and hence has the chemical formula C 6H12O6. Many organisms also synthesize other monosaccharides that have the same chemical formula as glucose but have different structures. These include fructose and galactose. Fructose is found in fruit. 19 Biomolecules: Basic molecules and enzymes Dr M. Ellul Glucose, fructose and galactose are isomers. Ring structures In solution, it is possible for sugars with 5 and 6 carbon atoms to form stable ring structures. In pentoses, the first carbon atom joins with the oxygen atom on the fourth carbon atom to give a furanose ring. In hexoses which are aldoses, for example glucose, the first carbon atom combines with the oxygen atom on carbon five to give a 6-membered ring – a pyranose ring. When glucose forms a ring structure, it can occur in two different forms with different properties: α-glucose and β-glucose. These structures differ only in the position of the —OH bound to carbon 1. When the hydroxyl is down, glucose is said to be in its α-form, and when it is up, glucose is said to be in its β-form. 20 Biomolecules: Basic molecules and enzymes Dr M. Ellul Draw the ring structure of α-glucose. How does β-glucose differ from it? Disaccharides Disaccharides are formed by condensation reactions between two monosaccharides. The bond formed between 2 monosaccharides is called a glycosidic bond and it normally forms between carbon atoms 1 and 4 of neighbouring units (a 1,4 bond). The most common disaccharides are maltose, lactose and sucrose: Maltose = Glucose + glucose 21 Biomolecules: Basic molecules and enzymes Dr M. Ellul Maltose is formed during digestion of starch. It is converted to glucose by the action of a maltase. The bond between two glucose residues is an 1-4 glycosidic linkage between C1 of a glucose molecule and C4 of a second glucose molecule. Lactose = glucose + galactose Lactose or milk sugar is found in milk. The bond between the glucose and galactose is a β 1-4 glycosidic linkage between C1 of a galactose molecule and C4 of a glucose molecule. Sucrose = glucose + fructose Sucrose, or cane sugar, is most abundant in plants, where it is translocated in large quantities through phloem tissue. It consists of an  1-2 glycosidic linkage between C1 of glucose and C2 of fructose. Sucrose is a non-reducing sugar. Oligosaccharides Oligosaccharides contain more than two monosaccharides. Glycoproteins and glycolipids found on the outer surface of cells have oligosaccharides attached to the R group of certain amino acids or lipids respectively. The human ABO blood types owe their specificity to oligosaccharides chains. Polysaccharides Polysaccharides are macromolecules that are polymers formed by many monosaccharides joined by glycosidic bonds in a condensation reaction to form chains. These chains may be: Branched or unbranched Folded (making the molecule compact which is ideal for storage, eg. starch and glycogen) 22 Biomolecules: Basic molecules and enzymes Dr M. Ellul Straight (making the molecules suitable to construct cellular structures, eg. cellulose) or coiled. Polysaccharides are insoluble in water. Polysaccharides function chiefly as food and energy stores and as structural materials. Starch Starch is a polymer of glucose. It is a major fuel store in plants, but it is absent from animals. Starch molecules accumulate to form starch grains. Starch is found in storage organs such as the potato tuber, and in seeds of cereals and legumes. Starch has two components, amylose and amylopectin. Amylose is composed of several α-glucose molecules linked together in long, unbranched chains. Each linkage occurs between the carbon 1 (C- 1) of one glucose molecule and the C-4 of another, making them α-(1, 4) glycosidic linkages Amylose is insoluble because the long chains of amylose tend to coil up in water. Potato starch is approximately 20% amylose. Amylopectin which makes up 80% of potato starch, is branched. The branches occur due to bonds between the C-1 of one molecule and the C-6 of another forming α-(1, 6) linkages. 23 Biomolecules: Basic molecules and enzymes Dr M. Ellul Plant cells store starch in specialised organelles known as amyloplasts. Plant cells store starch rather than glucose because: 1. Starch is a large, insoluble polysaccharide that does not dissolve in water, meaning it does not contribute to the osmotic pressure inside the cell. 2. Starch, being a branched/ coiled polymer of glucose, forms a compact way to store glucose molecules. A large number of glucose molecules can be packed into a single starch granule, which makes it an efficient storage form. Starch can be broken down slowly into glucose as needed, providing a steady supply of energy. Glycogen Glycogen is a storage polysaccharide made from glucose in animals and in fungi. In vertebrates, glycogen is stored chiefly in the liver and muscles. Glycogen is more branched than amylopectin and therefore it more compact which helps animals store more (more efficient as a storage polysaccharide molecule). Since there is more branching there are more free ends where glucose molecules can either be added or removed. This ensures that condensation and hydrolysis reactions occur rapidly – thus the storage or release of glucose can suit the demands of the cell. 24 Biomolecules: Basic molecules and enzymes Dr M. Ellul Cellulose Cellulose is another polymer of glucose. It is a structural component of all plant cell walls. It consists of long chains of β-glucose joined together by 1,4 glycosidic bonds. As β-glucose is an isomer of α- glucose to form the 1,4 glycosidic bonds, consecutive β-glucose molecules must be rotated 180° to each other. Due to the inversion of the β-glucose molecules there is the formation of many hydrogen bonds between the long chains giving cellulose its strength. Cellulose is the main structural component of plant cell walls due to its strength which is a result of the many hydrogen bonds found between the parallel chains of microfibrils. The high tensile strength of cellulose allows it to be stretched without breaking which makes it possible for cell walls to withstand turgor pressure. The cellulose fibres and other molecules (eg. lignin) found in the cell wall form a matrix which increases the strength of the cell walls. The strengthened cell 25 Biomolecules: Basic molecules and enzymes Dr M. Ellul walls provide support to the plant. Cellulose fibres are freely permeable which allows water and solutes to leave or reach the cell surface membrane. As few organisms have the enzyme (cellulase) to hydrolyse cellulose, it is a source of fibre. Comparison of polysaccharides How do the structures of starch, glycogen, and cellulose affect their function? _____________________________________________________________________________________________ _____________________________________________________________________________________________ _____________________________________________________________________________________________ _____________________________________________________________________________________________ _____________________________________________________________________________________________ _____________________________________________________________________________________________ _____________________________________________________________________________________________ _____________________________________________________________________________________________ _____________________________________________________________________________________________ 26 _____________________________________________________________________________________________ _____________________________________________________________________________________________ Biomolecules: Basic molecules and enzymes Dr M. Ellul Compounds closely related to polysaccharides Chitin Chitin is a structural material found in the exoskeleton of arthropods and in the cell wall of many fungi. It is a polymer of N-acetylglucosamine which is a derivative of glucose that contains nitrogen. When cross-linked by proteins, it forms a tough, resistant surface material that serves as the hard exoskeleton of insects and crustaceans. Murein The cell wall of bacteria is made of murein which is also known as peptidoglycan. Murein is a molecule formed from carbohydrate polymers linked together by peptide cross-bridges. Self-assessment 1. How do disaccharides form? 2. What type of bond that forms between two monosaccharides? 3. What are polysaccharides? What are their monomers? 4. What important roles do polysaccharides play in animals? Plants? Reflect on what you learnt: Distinguish between monosaccharides, disaccharides and polysaccharides. 27 Biomolecules: Basic molecules and enzymes Dr M. Ellul Practical Aspects Tests for sugars There are two types of sugars we can test for: reducing and non-reducing. Reducing sugars act as reducing agents in chemical reactions (i.e. donates electrons to other molecules). Reducing sugars include all monosaccharides (e.g. glucose and fructose) and some disaccharides (e.g. lactose and maltose). Non-Reducing sugars do not act as reducing agents in chemical reactions. They include most disaccharides (e.g. sucrose) and polysaccharides. Benedict’s test: Benedict’s reagent is the solution used in Benedict’s test. It is a bright blue solution containing: copper sulfate pentahydrate, sodium citrate sodium carbonate What is happening during the reaction? Benedict’s test is performed by heating the reducing sugar with Benedict‘s reagent. The presence of the alkaline sodium carbonate converts the sugar into a strong reducing agent called enediols. During the reduction reaction, the mixture will change its colour from blue to brick-red precipitate due to the formation of copper (I) oxide (Cu2O). Cu2+ or copper (II) form is reduced to Cu+ or copper (I). The red-coloured copper (I) oxide is insoluble in water and hence forms a precipitate. If the concentration of the sugar is high, then the colour becomes more reddish, and the volume of the precipitate increases. Test: 1. 1 mL of sample is added to a test-tube and 1 mL of Benedict’s solution is added to it. 2. The test-tube is heated in a boiling water bath for approximately 1 minute until a colour change is observed. 3. If a reducing sugar is present, a coloured precipitate is present. The colour of the precipitate depends on the concentration of the reducing sugar. If no reducing sugars are present, the solution remains blue. 28 Biomolecules: Basic molecules and enzymes Dr M. Ellul To test for non-reducing sugars: Test only done on a negative Benedict’s test for reducing sugar. 1. 1 ml of sample is added to a new test-tube. 1 ml Hydrochloric acid is added and the test-tube is heated in a boiling water bath for 1 minute. (To break the glycosidic bonds). 2. Sodium hydrogencarbonate is added to the test-tube until there is no more effervescence. (To neutralise the excess acid). 3. 1 ml of Benedict’s solution is added to the test-tube. 4. The test-tube is heated in a boiling water bath for approximately 1 minute until a colour change is observed. 5. If coloured precipitate forms, non-reducing sugars are present. There are other tests for reducing sugars: Fehling’s test: Test for starch: Add iodine in potassium iodide solution (Lugol’s iodine) to sample. If starch is present: it goes from orange/brown to blue-black. The basic principle involved in the iodine test is that amylose interacts with starch to form a blue-black coloured complex with the iodine. Iodine in potassium iodide forms polyiodide ions (triiodide, pentaiodide). These polyiodide ions form a blue-black complex with amylose. 29 Biomolecules: Basic molecules and enzymes Dr M. Ellul 30 Biomolecules: Basic molecules and enzymes Dr M. Ellul 2.1.3: Lipids Syllabus: Formation of triglycerides from alkanoic acids (fatty acids) and propane-1,2,3 triol (glycerol). Their main role as energy stores. Phospholipids: hydrophilic and hydrophobic properties in formation of membranes. Steroids: cholesterol, steroid hormones and Vitamin D (Detailed structure is not required but only the skeleton of a steroid as a set of complex rings of carbon atoms.) 31 Biomolecules: Basic molecules and enzymes Dr M. Ellul Learning outcomes Describe the structure of triglycerides. Explain how fats function as energy-storage molecules. Describe the structure of phospholipids. Apply knowledge of the structure of phospholipids to the formation of membranes. Describe the structure of steroids as being composed of complex rings of carbon atoms. Lipids are a diverse assortment of molecules, all of which share two important features: Lipids contain large regions composed almost entirely of hydrogen and carbons, with nonpolar carbon-carbon or carbon-hydrogen bonds. They lack any polar groups and therefore lipids hydrophobic and insoluble in water. Animal fats, oils, phospholipids and steroids are all lipids. Some lipids are energy storage molecules; some form waterproof coverings on both plant and animal bodies; some make up the bulk of all of the membranes of a cell; others are hormones. Lipids cannot be defined precisely because their chemistry is so variable, but we could say that true lipids are esters of fatty acids and an alcohol. Esters are organic compounds formed by a reaction between an acid and an alcohol: Acid + Alcohol → ester + water CH3COOH + C2H5OH → CH3COOC2H5 + H2O -COO- is an ester linkage. Many lipids are made up of two main kinds of molecules: fatty acids and glycerol. Fatty acids Fatty acids (also known as alkanoic acids) contain the acidic group —COOH (the carboxyl group) and are so named because some of the larger molecules in the series occur in fats. They have the general formula R.COOH where R is long hydrocarbon chain with many carbon atoms. Most fatty acids have an even number of carbon atoms between 14 and 22 per molecule. The hydrocarbon chain is non-polar and determines many of the properties of lipids. Being non-polar, the hydrocarbon chains are hydrophobic. Fatty acids can be saturated or unsaturated. 32 Biomolecules: Basic molecules and enzymes Dr M. Ellul Saturated fatty acids have no double bonds between the carbon atoms. Fatty acids sometimes contain one or more double bonds (C=C), such as oleic acid. They are said to be unsaturated. Unsaturated fatty acids melt at a much lower temperatures than saturated fatty acids. The fatty acid will have a kink in its hydrocarbon chain wherever a cis double bond occurs. (The double bonds in unsaturated fatty acids can exist in either a cis or a trans configuration. This describes whether the hydrogen atom is on the same side (cis) or opposite sides (trans). A cis double bond generates a bend in the molecule, influencing its structure and downstream function. Trans fats are rare in nature.) Unsaturated fatty acids with one double bond in its hydrocarbon chain is known as monounsaturated. Polyunsaturated fatty acids have from 2 to 6 double bonds in the hydrocarbon chain e.g. omega-3 fats. Glycerol Glycerol (also known as propane-1,2,3 triol) is an alcohol with three hydroxyl (-OH) groups, all of which can condense with a fatty acid to form an ester. Triglycerides Usually, all three undergo condensation and the lipid formed is called a triglyceride. 33 Biomolecules: Basic molecules and enzymes Dr M. Ellul Triglycerides are the commonest lipids in nature and if they are solid at 20°C they are called fats and if liquid they are oils. They are non-polar and therefore relatively insoluble in water. They are less dense than water and therefore they float. A major function of triglycerides is to act as energy stores. Triglyceride breakdown yields more energy than the breakdown of carbohydrates because the carbons are all bonded to hydrogens (and they, therefore, have a higher proportion of hydrogens relative to oxygens). This means they are electron- rich and can contribute to the production of acetyl-CoA, which is an important co-enzyme in aerobic respiration. Triglycerides contain more energy per gram than polysaccharides. Mammals store extra fats when hibernating, and fat is also found below the dermis of the skin in the form of adipose tissue where it serves as an insulator (mammals in cold climates; blubber in aquatic mammals where it contributes to buoyancy). Fats also form a protective layer around organs. When fats are oxidised, water is a product. This metabolic water can be very useful to some desert animals, such as the kangaroo rat. Plants usually store oils rather than fats. Seeds, fruits and chloroplasts are often rich in oils and some seeds are commercial sources of oils (e.g. coconut, soyabean). 34 Biomolecules: Basic molecules and enzymes Dr M. Ellul Phospholipids The following molecule is a phospholipid: Consider the term phospholipid: a. What portion of the molecule is responsible for the “phospho-” part of the name? b. What portion of the molecule is responsible for the “-lipid” part of the name? c. Circle the portion of the molecule that is polar. d. Would this portion of the phospholipid mix well with water? ____________________ e. Draw a square around the portion of the molecule that is nonpolar. f. Would this portion of the phospholipid mix well with water?____________________ Adapted from: POGIL™ Activities for AP* Biology 35 Homeostasis and Hormonal control Dr M. Ellul Phospholipids are lipids containing a phosphate group. The commonest type, phosphoglycerides, are formed when one of the primary alcohol groups (CH2OH) of glycerol forms an ester with phosphoric acid (H3P04) instead of a fatty acid. The molecule consists of a phosphate head with 2 hydrocarbon tails (the fatty acids). The phosphate group usually has a charged organic molecule linked to it, e.g. choline. (Image source: Raven et al. (2023)) Fatty acid chains are non-polar and are therefore hydrophobic. Phosphate group is polar and is hydrophilic. Therefore, a phospholipid is an amphipathic molecule which means it has both a hydrophobic and a hydrophilic component. In water, phospholipid molecules arrange themselves into a bilayer with the hydrophilic heads facing outwards into the water and hydrophobic tails facing inwards, avoiding water. This is the basic structure of a cell membrane. Why do phospholipids form membranes while triglycerides form insoluble droplets? ________________________________________________________________________________________ ________________________________________________________________________________________ ________________________________________________________________________________________ ________________________________________________________________________________________ 36 ________________________________________________________________________________________ Homeostasis and Hormonal control Dr M. Ellul Steroids Steroids are structurally different from all the other lipids. All steroids are composed of four rings of carbon fused together, with various functional groups protruding from them. Steroids include cholesterol, which is a vital component of the membranes of all eukaryotic cells and which is used by cells to synthesize other steroids. These other steroids include male and female sex hormones e.g. testosterone and vitamin D. Self-assessment 1. What are the components of triglycerides? 2. Describe the formation of triglycerides from glycerol and fatty acids. 3. Draw a phospholipid. Label all parts, including polar and nonpolar regions. 4. Phospholipids play a major role in cells. Where can they be found in a cell, and what is their role? 5. What are steroids? Give the role of some important steroids. Reflect on what you learnt: Draw a concept map summarising the topic ‘lipids’ 37 Homeostasis and Hormonal control Dr M. Ellul Practical aspects Tests for lipids Emulsion test 1. Dissolve the substance by mixing it with absolute ethanol. 2. Decant the ethanol into water. 3. If lipids are present in the mixture, it will precipitates and forms an milky emulsion. Because lipids are insoluble in water, they are immiscible with the water. Consequently, any lipids present in the sample will float to the top and form a milky white emulsion. Sudan (III) test Sudan III is a red fat-soluble dye that is utilized in the identification of the presence of lipids, triglycerides and lipoproteins. The oil will stain red with Sudan III dye since it is a lipid and contains triglycerides. However, since the oil is less dense than water and insoluble in water, the oil will form a layer or globules above the water and appear as a red layer above the water in the test tube. 38 Homeostasis and Hormonal control Dr M. Ellul 2.1.4: Proteins Syllabus: General structure of an amino acid to include different properties of R groups and cysteine and methionine as examples of S- containing R groups; peptide linkage; primary structure; secondary structure to include α-helix and β-pleated sheet; tertiary structure involving H-bonding, ionic bonds, disulphide bridges and hydrophobic and hydrophilic interactions; quaternary structures of proteins. Importance of shape in protein function: fibrous proteins have a structural role e.g. collagen; globular proteins mostly function as enzymes, antibodies and hormones e.g. insulin. 39 Homeostasis and Hormonal control Dr M. Ellul Learning outcomes Describe the structure of amino acids. Explain the formation of peptide bonds. Describe the different levels of protein structure. Explain the importance of shape in protein function. Proteins are very important macromolecules and account for over 50% of the dry mass in most cells. Proteins serve numerous critical functions including: acting as enzymes forming integral components of cell membranes hormones oxygen carrying pigments like haemoglobin and myoglobin antibodies providing strength to animal tissues, such as bone and arterial walls through proteins like collagen structural proteins that is found in hair, nails and skin’s surface layers e.g. keratin enabling muscle contraction through actin and myosin proteins. Proteins are polymers and the monomers building up the proteins are known as amino acids. Amino acids Using the table of functional groups on pg 15 of the notes: 1. a. Draw a circle around the amino group in the diagram. b. Draw a triangle around the carboxylic acid (carboxyl) group. 2. How are the amino acids similar to one another? _________________________________________________________________________________ _____________________________________________________________________________ 3. How are the amino acids different from one another? __________________________________________________________________________ __________________________________________________________________________ Adapted from: POGIL™ Activities for AP* Biology 40 Homeostasis and Hormonal control Dr M. Ellul The general formula of an amino acid is: H R C COOH NH2 Plants are able to make all the amino acids that they required from simpler substances. However, animals are unable to synthesize all that they need, and therefore must obtain some ‘ready-made’ amino acids directly from their diet. These are termed essential amino acids. Structure and range of amino acids Except for proline and hydroxyproline (which are imino acids =NH instead of –NH2), all amino acids are  amino acids, that is the amino group (-NH2) is attached to the  carbon of the related carboxylic (-COOH) group. The majority of amino acids possess one acidic carboxylic group and one basic amino group and are termed “neutral” amino acids. When there are more than one amino group present it is called basic amino acids; when there is more than one carboxylic acid it is known as acidic amino acids. The R group represents the residual part of the molecule. The simplest amino acid, glycine is formed when R is substituted by H. When the R is anything but H, the groups attached to the  carbon will all be different. The carbon is therefore asymmetric. The amino acid will exhibit optical isomerism. The R group determines the characteristics of the amino acid. When R is substituted by –CH3, the amino acid alanine is formed. Two amino acids contain sulfur. These are methionine and cysteine. Methionine is non-polar and encoded solely by the AUG codon. It is the “initiator” amino acid in protein synthesis, being the first one incorporated into protein chains. Properties of amino acids Amino acids are colourless, crystalline solids. They are generally soluble in water but insoluble in organic solvents. 41  Homeostasis and Hormonal control Dr M. Ellul In neutral aqueous solutions, they exist as dipolar ions (zwitterions) and are amphoteric, possessing both basic and acidic properties. Each amino acid has its own specific pH at which it will exist in its neutral zwitterion form and will be strongly dipolar. If it is placed in an electric field at this pH, it will not migrate neither to the cathode nor to the anode. The pH causing this electric neutrality is called the isoelectic point of the amino acid. Solution made Solution made more acidic. more basic. H+ ions H+ ions accepted. The donated. The amino acid amino acid becomes becomes positively negatively charged charged Chemical bonds Amino acids are able to form a variety of chemical bonds with other reactive groups. Peptide bond This is formed when a water molecule is eliminated during interaction between the amino group of one amino acid and the carboxylic group of another. Elimination of water is known as condensation and the linkage formed is a covalent C-N bond called peptide bond. The compound formed is a dipeptide. If many amino acids are joined together in this way a polypeptide is formed. 42 Homeostasis and Hormonal control Dr M. Ellul a. How many amino acids are involved in the reaction to make a dipeptide?_______________ b. In the diagram above, the original amino acids are combined through a condensation reaction to make the dipeptide. i. What does R1 represent in the dipeptide? _____________ ii. What does R2 represent in the dipeptide?_____________ c. Put a box around the atoms in the amino acids that become the H 2O molecule produced by the reaction. d. A peptide bond is a covalent bond linking two amino acids together in a peptide. Circle the peptide bond. e. Between which two atoms in the dipeptide is the peptide bond located? ______________ f. Between what two functional groups is the peptide bond located? _________________________________ Adapted from: POGIL™ Activities for AP* Biology 43 Homeostasis and Hormonal control Dr M. Ellul Proteins Proteins are complex organic compounds always containing the elements carbon, hydrogen, oxygen and nitrogen and in some cases sulphur. Some proteins form compounds with other molecules containing phosphorus, iron, zinc and copper. Proteins are macromolecules of high relative molecular mass, between several thousands and several millions, consisting of chains of amino acids. 20 different amino acids are commonly found in naturally occurring proteins. Proteins are the most abundant organic molecules to be found in cells and comprise over 50% of their total dry mass. They are an essential component of diet in animals. Structure of proteins Structure of proteins Primary structure The primary structure is the number and sequence of amino acids held together by peptide bonds in a polypeptide chain. The sequence is coded by the genes. Different types of proteins have different sequences of amino acids. 44 Homeostasis and Hormonal control Dr M. Ellul a. Draw an arrow to two different peptide bonds in the diagram. b. Circle three separate amino acids that were joined together to make the polypeptide. Adapted from: POGIL™ Activities for AP* Biology Secondary structure What types of bonds are holding the secondary structure in place? ______________________ What groups on the amino acids are always involved in these bonds? _____________________ Draw a rectangle around two different R groups on the amino acids in the secondary structure. Is there any interaction between R groups in the secondary structure? ____________________ Adapted from: POGIL™ Activities for AP* Biology 45 Homeostasis and Hormonal control Dr M. Ellul Hydrogen bonds cause may protein chains to form secondary structures. In a peptide, every amino acid retains a –C=O from its carboxyl group and N-H from its amino group. Because oxygen attracts more electrons than carbon, it is relatively negative. Similarly, N attracts electrons more strongly than hydrogen and leaves hydrogen more positive. Therefore, hydrogen bonds can form between the oxygen of the –C=O and the hydrogen of the –N-H. Many proteins have a coiled springlike shape called α helix in which the hydrogen bonds hold the turns of the coil together. An example of such a protein is keratin. Another type of secondary structure is the -pleated sheet. Some proteins, for example silk, consist of many protein chains lying side by side, with hydrogen bonds holding adjacent molecules in a pleated sheet arrangement. Which molecule represents an α-helix? __________________________ Which molecule represents a β-pleated sheet? ____________________ Adapted from: POGIL™ Activities for AP* Biology 46 Homeostasis and Hormonal control Dr M. Ellul These secondary structures can again fold into something called super-secondary structures or motifs. These are specific combinations/folding patterns that consists of normal secondary structures: (Source: Raven et al., 2023) Tertiary structure Usually the polypeptide chain bends and folds extensively forming a ‘globular’ shape. This is maintained by the interaction of: Hydrogen bonds (these are between R groups) Disulphide bridges (only occurs between cysteine amino acids) Ionic bonds (occurs between charged R groups) Weak hydrophobic (between non-polar R groups; also known as van der Waals interactions) and hydrophilic interactions The tertiary structure of a protein can be determined by X-ray crystallography. 47 Homeostasis and Hormonal control Dr M. Ellul a. Four types of bonds or interactions are shown. Label them with the following terms: Disulfide bridge Hydrogen bond Hydrophobic interactions Ionic bond b. Describe the part of the amino acid that participates in these interactions. _______________________________________________________________________________ c. How does your answer in part b differ from the bonds that stabilize the secondary structure? _______________________________________________________________________________ d. What type of functional groups or atoms would need to be present in the R-groups for hydrogen bonding to occur between two amino acids in a protein chain? _______________________________________________________________________________ e. What type of functional groups or atoms would need to be present in the R-groups for hydrophobic interactions to occur between two amino acids in a protein chain? ______________________________________________________________________________ f. How many polypeptide chains are shown in the tertiary protein structure? ______________________________________________________________________________ Adapted from: POGIL™ Activities for AP* Biology 48  Quaternary structure a. How many polypeptide chains are shown in the quaternary structure of the protein in the diagram above? _____________ b. What types of bonds and interactions hold the quaternary structure in place? ______________________________________________________________________ ______________________________________________________________________ Adapted from: POGIL™ Activities for AP* Biology Many highly complex proteins consist of an aggregation of polypeptide chains held together by hydrophobic interactions, hydrogen and ionic bonds and disulfide bridges. Their arrangement is of the quaternary structure. Haemoglobin exhibits such a structure. It consists of 4 separate polypeptide chains of two types, namely 2  - chains and 2  - chains. The 2  - chains contain 141 amino acids. The 2  - chains contain 146 amino acids. Haemoglobin is the oxygen-carrying protein found in animals. It is a globular protein that belongs to the heme protein family. In such globular proteins, the haem group is strongly linked to the protein structure. The heme group's function depends on the protein's structure. The haem group in haemoglobin binds oxygen molecules. Haem is the most important part of haemoglobin. It is complex containing iron (II) ion (Fe2+) at its centre. This Fe2+ ion can form two additional bonds, one with the oxygen molecule and one with the amino acid.  Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Haemoglobin molecule Haem group Review (Source: Raven et al., 2023) 50 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Level of structure Description Types of Bond Primary Secondary Tertiary Quaternary 51 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Denaturation of proteins Denaturation is the loss of the specific 3-dimentional conformation of proteins. It is caused by: Heat Strong acids and alkali and high concentrations of salts. Heavy metals Organic solvents and detergents. The importance of shape in protein function Proteins are intricate macromolecules composed of amino acids folded into specific three- dimensional structures, and this structural arrangement directly governs their functions. Fibrous proteins, e.g. collagen, play a fundamental structural role in the body. Collagen has a unique triple- helix structure forms a sturdy, fibrous framework that provides tensile strength to tissues such as skin, tendons, and bones. Its elongated, rope-like shape enables it to resist stretching forces, ensuring the integrity and resilience of connective tissues. In contrast, globular proteins, primarily function as enzymes, antibodies, and hormones. The compact, spherical shape of globular proteins facilitates their specific interactions with target molecules. Enzymes, for instance, act as biological catalysts by binding substrates within their active sites, promoting chemical reactions. Antibodies adopt a globular shape to recognize and neutralize foreign invaders, such as bacteria and viruses, while hormones like insulin regulate glucose levels in the bloodstream by binding to receptors on target cells. Fibrous proteins Globular proteins offer structural support and stability due to Their precisely folded shapes allow them to their elongated, fibrous structure, enabling recognize specific substrates, antigens, or them to endure mechanical stress and receptors, leading to finely tuned maintain tissue integrity biochemical reactions and immune responses Self-assessment 1. How many monomers of proteins are there? 2. Draw the general structure of an amino acid. 3. How do the R groups of amino acids contribute to protein structure? 4. True/false: a change in a protein’s structure will change the protein’s function? 5. How does the primary structure of a protein affect the other structural levels? 6. Would the function of a protein change if the amino acid sequence changed? Why or why not? 7. What interactions occur in the secondary structure? Tertiary structure? Quaternary structure? 8. What causes a protein to denature? What happens to a protein if it is denatured? 52 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Reflect on what you learnt: 1. What groups do all amino acid 2. Describe how a dipeptide is formed. molecules have in common? 3. Briefly describe the different structural levels of proteins 53  Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Practical aspect Biuret Test - Test for Proteins The Biuret test can be used to test for the presence of peptide bonds in a protein and therefore to detect proteins in food. The test is carried out as follows: 1. Place the sample to be tested in a test tube and add an equal volume of sodium hydroxide at room temperature. 2. Add a few drops of very dilute (0.05%) copper (II) sulfate soliton and mix gently. 3. A purple colouration indicates the presence of a peptide bond and hence a protein. A negative result would mean the solution remains blue. iew Rev 54 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul 2.2: Enzymes 2.2.1: Organic Catalysts 2.2.2: Site of enzymes Syllabus: Enzyme structure and function; Energy changes in chemical reactions and activation energy: lowering of activation energy through the formation of an enzyme-substrate complex. Site of enzymes: In solution and as part of cell membranes or organelle membranes. 55 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Learning outcomes Describe the structure of enzymes. List the properties of enzymes. Describe the mechanisms of how enzymes work. Enzymes are protein molecules produced by living cells. Enzymes can be defined as biological catalysts, that is, they speed up the rate of metabolic reactions. They are vitally important because in their absence, reactions in the cells would be too slow to sustain life. Properties of enzymes Biological reactions Energy releasing processes, ones that "generate" energy, are termed exergonic reactions. Reactions that require energy to initiate the reaction are known as endergonic reactions. 56 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Enzyme reactions may be either: Catabolic - involved in breakdown. Anabolic - involved in synthesis. Metabolism consists of anabolism and catabolism. The energy required to make substances react is called the activation energy (EA). The greater the amount of activation energy required the slower will be rate of reaction at a given temperature. Enzymes serve to reduce the activation energy required for a chemical reaction to take place. Enzymes are biological catalysts that speed up the rate of metabolic reactions by reducing the activation energy. 57 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Mechanisms of enzyme action An enzyme combines with its substrate to form a short-lived enzyme-substrate complex. Within this complex the chances of reactions occurring are greatly enhanced. Once a reaction has occurred, the complex breaks up into products and enzymes. The enzyme remains unchanged at the end to the reaction and is free to interact again with the substrate. Most enzymes are much larger than the substrate on which they react. In fact, only a very small portion of the enzyme, between 3-12 amino acids, comes in direct contact with the substrate. This region is called the active site of the enzyme. Lock and key hypothesis – suggested by Fischer (1890) The enzyme had a particular shape into which the substrate or substrates fit exactly. The substrate is the key whose shape is complementary to the enzyme or lock. When an enzyme/substrate complex is formed, it is activated into forming the products of the reaction. Once formed, the products no longer fit into the active site and escape to the surrounding medium, leaving the active site free to receive further substrate molecules. 58 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Induced fit hypothesis – suggested by Koshland (1959) When a substrate combines with an enzyme, it induces changes in the enzyme structure. The amino acids, which constitute the active site, are shaped into a precise formation that enables to perform its catalytic function most effectively. 59 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Learning outcomes Identify that enzymes can catalyse reactions both intracellularly and extracellularly. Where are enzymes found? Enzymes can catalyse biochemical reactions both in the cytoplasm or organelles within the cell as well as in the extracellular environment. Enzymes working in both these environments are essentially synthesized by ribosomes. Intracellular enzymes are present inside the cell membrane. Intracellular enzymes may be present freely in the cytoplasmic fluid of the cell or they may be bound to some organelles such as ribosomes. Enzymes may also present within the membrane- bound organelles within the cell such as mitochondria, lysosomes, nucleus, etc. Enzymes can also be found within the cell membrane e.g. ATPase. Cytoplasmic enzymes The cytoplasm is the main hub of cellular metabolism. The majority of metabolic processes take place within the cytoplasm of cells. The important intracellular enzymes that catalyse the following metabolic processes work within the cytoplasm: glycolysis gluconeogenesis PS these processes will be done in detail later glycogen metabolism on during the course urea cycle amino acid metabolism Extracellular enzymes The extracellular enzymes are present outside the cells, in the extracellular fluid. They are present in tissue spaces, body fluids, and organ cavities. Some examples of extracellular enzymes are salivary amylase, pepsin, chymotrypsin, elastases, collagenases, pancreatic amylase, pancreatic nucleases, and nucleosidases, etc. Moreover, intestinal enzymes such as peptidase, sucrase, and maltase are also extracellular enzymes. Reflect on what you learnt: Are digestive enzymes extracellular or cytoplasmic enzymes? 60 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul 2.2.3: Factors affecting the rate of enzyme-catalysed reactions Syllabus: Temperature, pH, enzyme and substrate concentration. 61 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Learning outcomes List and explain the factors that influence the rate of enzyme-catalysed reactions. Rate of enzyme reactions The rate of an enzyme reaction is measured by the amount of substrate changed or amount of product formed over a period of time. Factors affecting the rate of enzyme-controlled reactions When investigating the effect of one factor on the rate of an enzyme-controlled reaction, all other factors should be kept constant and at optimum level. Enzyme concentration The higher the enzyme concentration in a reaction mixture, the greater the number of active sites available and the higher the probability of enzyme-substrate complex formation. The rate of reaction increases linearly with enzyme concentration. (If the amount of substrate is limited, at a certain point any further increase in enzyme concentration will not increase the reaction rate as the amount of substrate becomes a limiting factor.) Substrate concentration For a given enzyme concentration, the rate of reaction increases with increasing substrate concentration until saturation point is reached, VMAX. This saturation point is theoretical. At this point, all the active sites eventually become filled, reaching saturation. Consequently, further increments in substrate concentration won't lead to a higher reaction rate and the graph plateaus. When all the enzyme's active sites are occupied, additional substrate molecules lack available binding sites to form enzyme-substrate complexes. 62 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Temperature Enzymes have an optimum temperature at which they catalyse a reaction at the maximum rate. Lower temperatures either prevent reactions from proceeding or slow them down because molecules have less kinetic energy resulting in a lower frequency of successful collisions between substrate molecules and the active sites of the enzymes which leads to less frequent enzyme- substrate complex formation. Substrates and enzymes also collide with less energy, making it less likely for bonds to be formed or broken. Higher temperatures cause reactions to speed up as molecules have more KE resulting in a higher rate of successful collisions between substrate molecules and the active sites of the enzymes leading to more frequent enzyme- substrate complex formation. Substrate and enzyme molecules also collide with more energy, making it more likely for bonds to be formed or broken. If temperatures continue to increase past the optimum temperature, the rate of reaction drops sharply, as the enzyme begins to denature. The increased KE and vibration of the enzyme molecules puts a strain on them, eventually causing the weaker hydrogen and ionic bonds that hold the enzyme molecule in its precise shape to start to break. The breaking of bonds causes the tertiary structure of the enzyme to change and the active site is permanently damaged and its shape is no longer complementary to the substrate, preventing the substrate from binding. Temperature coefficient Over the temperature The effect of temperature on the rate of a reaction range 0-40oC, Q10 for an can be expressed as temperature coefficient Q10 enzyme-controlled reaction is 2. For every rise rate of reaction at (x + 10) °C of 10oC, the rate of an Q10 = enzyme-controlled rate of reaction at x °C reaction is doubled. 63 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul pH All enzymes have an optimum pH or a pH at which they operate best. Enzymes are denatured at extremes of pH. Below and above the optimum pH of an enzyme, solutions with an excess of H+ ions (acidic solutions) and OH- ions (alkaline solutions) can cause these hydrogen and ionic bonds to break. The breaking of bonds alters the shape of the active site, which means enzyme-substrate complexes form less easily. Eventually, enzyme-substrate complexes can no longer form at all. At this point, complete denaturation of the enzyme has occurred. Where an enzyme functions can be an indicator of its optimal environment: Eg. pepsin is found in the stomach, an acidic environment at pH 2 (due to the presence of hydrochloric acid in the stomach’s gastric juice). Pepsin’s optimum pH is pH 2. Reflect on what you learnt: Draw annotated graphs representing the factors affecting the rate of enzyme controlled reactions. 64 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul 2.2.4: Enzyme inhibition 65 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Learning outcomes Distinguish between competitive and non-competitive inhibition. Interpret graphs depicting competitive and non-competitive inhibition. A variety of small molecules exist which can reduce the rate of an enzyme-controlled reaction. These are called enzyme inhibitors. Inhibition can be of different types: Competitive Reversible Non-competitive Inhibition Irreversible End-product inhibition Reversible inhibition The inhibitor may be easily removed from the enzyme under certain conditions. Competitive Reversible inhibition Here a compound, structurally similar to that of the usual substrate associates with the enzyme active site but is unable to react with it. Example: 66 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Non-competitive reversible inhibition This type of inhibitor has no real structural similarity to the substrate and forms an enzyme- inhibitor complex at a point on the enzyme other than its active site. Irreversible inhibition Very small concentrations of chemical reagents e.g. heavy metal ions (Hg 2+; Ag+; As+(Arsenic)) completely inhibit some enzymes. They react covalently with the –SH groups and cause the protein of the enzyme molecule to precipitate. If these are components of the active site, then the enzyme is inhibited. Reflect on what you learnt: Differentiate between the different types of enzyme inhibition. 67 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul 2.2.5: Allosteric enzymes Syllabus: Syllabus Their role in regulating metabolic pathways by negative feedback inhibition as exemplified by phosphofructokinase. 68 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Learning outcomes Describe allosteric regulation. Explain end-product inhibition (negative feedback inhibition) In allosteric regulation, enzyme action is inhibited or enhanced when the molecules bind to a binding site on the enzyme that is distinct from the active site. This separate binding region is called the allosteric regulatory site. When the allosteric regulatory site is occupied, the enzyme changes shape and its activity may be inhibited or enhanced. End-product inhibition (negative feedback inhibition) When the end-product of a metabolic pathway begins to accumulate, it may act A metabolic as an allosteric inhibitor on the enzyme controlling the first step of the pathway. pathway is a linked series of reactions happening in a cell Example of negative feedback inhibition Glycolysis is a metabolic pathway forming the first part of cellular respiration. Several steps in glycolysis are regulated, but the most important control point is the third step of the pathway, which is catalyzed by an enzyme called phosphofructokinase (PFK). PFK is regulated by ATP and citrate. ATP: ATP is a negative inhibitor of PFK. If there is a lot of ATP in the cell, glycolysis does not need to make more. 69 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Citrate: Citrate is the first product of the next series of reactions in cellular respiration called the Kreb’s cycle. This also inhibits PFK. If citrate builds up, this is a sign that glycolysis can slow down, because the Kreb’s cycle has enough products coming from glycolysis. Reflect on what you learnt: Explain, in your own words, what is meant by allosteric regulation. 70 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul w vie Re 71 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul 2.1.6: Vitamins and their roles as co-enzymes Syllabus: NAD+/NADH; NADP+/NADPH; FAD/FADH2 and coenzyme A. 72 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Enzyme cofactors Learning outcomes Define the term coenzyme. Explain how NAD+/NADH; NADP+/NADPH; FAD/FADH2 work in terms of redox reactions. Explain the action of coenzyme A Many enzymes require non-protein components called cofactors for their efficient activity. Cofactors may vary from simple inorganic ions to complex organic molecules. The enzyme/cofactor complex is called a holoenzyme, whilst the enzyme portion without its cofactor is called apoenzyme. There are different types of cofactors e.g. coenzymes. Coenzymes Larger organic cofactors are known as coenzymes. The coenzymes are loosely associated with the enzyme. Coenzymes link different enzyme-catalysed reactions into a sequence during metabolic processes, such as photosynthesis and respiration. Coenzymes act as carriers of groups of atoms, single atoms or electrons that are being transferred from one place to another in an overall metabolic pathway. Vitamins are essential organic compounds that are required in small amounts by the body to support various biochemical processes and maintain overall health. Vitamins are an important source of coenzymes. For example, many vitamins in the B vitamin group are used in the production of important coenzymes, including: Pantothenic acid, a key component of coenzyme A (a coenzyme required for the oxidation of pyruvate during the link reaction that occurs between the glycolysis and Krebs cycle stages of respiration) Nicotinic acid (niacin), used to produce the coenzymes NAD+ and NADP (coenzymes required in many different metabolic reactions, including many of the reactions that take place during photosynthesis and respiration) 73 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Vitamin B2 (riboflavin), used to produce the coenzyme FAD (a coenzyme required in the Krebs cycle during respiration) Examples of coenzyme functions Coenzymes NAD+ (nicotinamide adenine dinucleotide) and flavin adenine dinucleotide (FAD) play an important role in aerobic respiration. NAD+/NADH NAD+ (Nicotinamide Adenine Dinucleotide): It plays a pivotal role Redox reactions: in redox reactions, e.g. during the transfer of electrons during Reduction-Oxidation reactions cellular respiration and fermentation. When hydrogen atoms become available at different points during respiration NAD+ Oxidation Reduction accepts these hydrogen atoms. Gain of Loss of oxygen oxygen A hydrogen atom consists of a hydrogen ion and an electron. Loss of Gain of hydrogen hydrogen When the coenzymes gain a hydrogen and two electrons, NAD+ is Loss of Gain of ‘reduced’ and becomes NADH. electrons electrons NADH (Reduced Nicotinamide Adenine Dinucleotide): NADH is the reduced form of NAD+. NADH transfers the hydrogen atoms (hydrogen ions and electrons) from the different stages of respiration to the electron transport chain on the inner mitochondrial membrane, the site where hydrogens are removed from the coenzymes. When the hydrogen atoms are removed the coenzyme is ‘oxidised’. Hydrogen ions and electrons are important in the electron transport chain at the end of respiration as they play a role in the synthesis of ATP. 74 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul (Source: Raven et al.) NADP+/NADPH NADP+ (Nicotinamide Adenine Dinucleotide Phosphate): NADP+ is a co-enzyme similar in structure to NAD+, but it is primarily involved in anabolic reactions, e.g. photosynthesis. NADPH (Reduced Nicotinamide Adenine Dinucleotide Phosphate): NADPH is the reduced form of NADP+. It acts as a reducing agent, by donating electrons. During photosynthesis NADPH brings about the reduction of carbon dioxide to carbohydrate as the metabolic reactions of photosynthesis proceed. FAD/FADH2 FAD (Flavin Adenine Dinucleotide): FAD is a co-enzyme derived from vitamin B2, also known as riboflavin. It functions as an electron carrier, participating in redox reactions. FAD accepts two electrons and two hydrogen ions. FADH2 (Reduced Flavin Adenine Dinucleotide): FADH2 is the reduced form of FAD. It carries electrons during biochemical reactions. 75 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul FAD and FADH2 are crucial for the electron transport chain in cellular respiration. Coenzyme A Coenzyme A acts in a different way, by transferring chemical groups. For example: coenzyme A is responsible for the transfer of an acetyl group (-CH₃CO) from fatty acids and glucose during respiration. It forms acetyl-CoA, a key molecule that enters the citric acid cycle for ATP production. Reflect on what you learnt: Briefly describe how the different coenzymes work. 76 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul 2.1.5: Nucleic Acids Syllabus: Nucleotides condense together by means of a phosphodiester bond to form a polynucleotide having a 5’end and a 3’end. 5 different nitrogenous bases: pyrimidines and purines. DNA: awareness that adenine and thymine have 2 hydrogen bonds and cytosine and guanine have 3 hydrogen bonds. The structures of DNA, mRNA and tRNA only in sufficient detail to provide an understanding of their roles in coding information and in protein synthesis. (Details of r-RNA structure is not required) 77 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Learning outcomes Define and describe nucleotides. Describe the formation of polynucleotide. Describe a DNA molecule. Describe mRNA and tRNA molecules. Nucleic acids are polymers composed of monomer units known as nucleotides. There are a very few different types of nucleotides. The main functions of nucleotides are: information storage (DNA – deoxyribonucleic acid); protein synthesis (RNA – ribonucleic acid), and energy transfers (ATP and NAD). Nucleotides consist of a sugar, a nitrogenous base a phosphate. The sugars are either ribose or deoxyribose. They differ by the lack of one oxygen in deoxyribose. Both are pentoses usually in a ring form. There are five nitrogenous bases which can occur in two structural forms: purines and pyrimidines. Adenine and Guanine are double-ring structures and are purines. Cytosine, Thymine and Uracil are single-ringed and are pyrimidines. Phosphate: this gives nucleic acids their acidic character. 78 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul When a sugar combines with a nitrogenous base, a compound called nucleoside forms. This occurs with the elimination of water (condensation reaction). A nucleotide is formed by further condensation between the nucleoside and phosphoric acid forming an ester bond. Two nucleotides join to form a dinucleotide by condensation between the phosphate group of one with the sugar of the other to form a phosphodiester bridge. The process is repeated up to several million times to make a polynucleotide. When nucleotides polymerise to form nucleic acids, the hydroxyl group attached to the 3’ carbon of a sugar of one nucleotide forms an ester bond to the phosphate of another nucleotide, eliminating a molecule of water. 79 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul The 3’ end has a free hydroxyl group attached to the 3’carbon of the sugar, the 5’ end has a phosphate group attached to the 5’ carbon of the sugar. Deoxyribonucleic acid (DNA) DNA consists of two strands, twisted about each other into a double helix. The sugar-phosphate backbone of the two DNA strands are on the outside of the double-helix. The bases are packed in the middle paired up to form the steps of the ladder. The two DNA strands are described as antiparallel. The first strand runs in 5’→ 3’ direction whilst the other strand runs in the 3’ → 5’ direction. (Phosphate group is found at the 5’, OH of sugar is found at the 3’.) Each step of the ladder is composed of a purine and a pyrimidine, held together by hydrogen bonds. Adenine and thymine are held together by two hydrogen bonds, cytosine and guanine are held together by three. In nucleic acids, bases that pair together by hydrogen bonds are called complementary base pairs. In DNA, adenine is complementary to thymine and guanine is 80 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul complementary to cytosine. This is called the base-pairing rule. Ribonucleic acid (RNA): RNA is ribonucleic acid. It is single stranded and have uracil as base instead of thymine present in DNA. Its nucleotide is formed from ribose sugar, nitrogenous base and phosphate. There are 3 main types of RNA: mRNA (messenger RNA) tRNA (transfer RNA) rRNA (ribosomal RNA) Messenger RNA (mRNA): It is made during transcription from DNA. It has single polynucleotide strand. Three adjacent bases form a group in mRNA, which is known as codons or triplets. Transfer RNA (tRNA): It is involved in translation. It is also single polynucleotide stranded but bends to form cloverleaf- like structure. This special shape is maintained by the hydrogen bonds between specific base pairs. They have specific triplets made at one end by specific bases, known as anticodon, which actually recognises the codon on mRNA. At another end they carry amino acids binding site where it carries amino acids through ribosomes according to the codons on mRNA. 81 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Compare and contrast DNA and RNA DNA RNA Nucleic Acids Similarities Macromolecules Structure Pentose sugar Differences Nitrogenous bases Types Location Function Passing to the next generation 82 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Self-assessment 1. Identify the three components of a nucleotide. 2. What is the monomer called if it is lacking a phosphate group? 3. List the possible nitrogenous bases that can be found in nucleotides. 4. What forms the “backbone” of DNA? Reflect on what you learnt: Draw a concept map summarising nucleic acids 83 Biochemistry (Basic Molecules and Enzymes) Dr M Ellul Putting it all together Macromolecule Elements Monomer Polymer Bond Involved between 2 monomers 84