Biochemistry

1. Biochemistry

2. Aim The aim of this option is to give you an understanding of the chemistry of important molecules in the human body, and the need for a balanced and healthy diet.

3. Energy

4. Contents Recall the dietary requirements of humans. Food calorimetry Calculate the energy value of a food from enthalpy of combustion data

5. Energy • Energy is provided by lipids (in fats), carbohydrates and proteins. • Carbohydrates provide the main source of energy but, like proteins, they are already partially oxidized, so do not provide as much energy weight for weight as fats, which are used to store energy. • The amount of energy in a dried food can be determined by combustion experiments using a food calorimeter.

6. Combustion calculations Energy Specific heat capacity Temperature change x mass x = ∆H = m x s x ∆T • Remember when we find energy we need to find energy per mole.

7. Proteins

8. Contents List the major functions of proteins in the body Draw the general formula of 2-amino acids. Describe the characteristic properties of 2-amino acids Describe the condensation reaction of 2-amino acids to form polypeptides

9. Proteins • Proteins are an essential part of a healthy diet. • Rich sources of protein include; meat, fish, cheeseeggs and nuts. • They have many different functions in the body • Biological catalysts (enzymes) • Transporters e.g. haemoglobin in the blood • Structure (e.g. hair and nails consist almost entirely of polypeptides coiled into proteins called keratin and composed of α-helices). • Some hormones are proteins or protein based, e.g. insulin and FSH (follicle stimulating hormone) responsible for triggering the monthly cycle in females.

10. What are they made of? • Proteins are large macromolecules made up of chains of 2-amino acids. • About 20 2-amino acids occur naturally, they have the general formula:H2NCHRCOOH. • Each 2-amino acid is given a three letter symbol, which is usually the first three letters of its name. Remember an R group can be any alkyl group H O H C N C H O H R Carboxylic acid group Amino group

11. Characteristic properties of amino acids • The structure of amino acids in aqueous solution alters at different pH values. • At low pH (acidic medium) the amine group will be protonated(gain a proton). • At high pH (alkaline medium) the carboxylic acid group will lose a proton. • For each amino acid there is a unique pH value, known as the isoelectric point, where the amino acid will exist as the zwitterion (when the amino group is protonated and the acid group loses an hydrogen)

12. 2-amino acids function as good buffers, because they can combine with either protons or hydroxide ions, thus helping to maintain the existing pH of the solution. • A buffer is a solution which resists changes in pH if smallamounts of acid or alkali are added to it.

13. Forming polypeptides • Two 2-amino acids can condense together to form a dipeptide with the elimination of water. • Two 2-amino acids can react differently to form two different dipeptides.

14. Peptide Bonds • The link between them, which has the structure: is known as a peptide bond. • The dipeptides still contain a reactive functional group on each end so they can react further in the presence of enzymes to form long chains of amino acid residues linked by peptide bonds. • This is an example of condensation polymerisationand the products are known as polypeptides O H N C

15. Condensation Polymerisation

16. Activity: • Draw the following dipeptides: • Ala – Arg • Arg – Ala • Glu – Gly • Leu – Lys • Phe - Ser

17. Contents Describe and explain the primary, secondary (α-helix and β-pleated sheets), tertiary and quaternary structure of proteins.

18. Structure of proteins There are four levels of structure to describe proteins; primary, secondary, tertiary and quaternary. • Primary structure – the amino acid sequence in the polypeptide chain • Secondary structure – the α-helix (intramolecular) and β-pleated sheets (intermolecular) held by hydrogen bonding. • Tertiary structure – overall folding of the protein molecule held by interactions between distant amino acids • Quaternary structure – fitting together two or more separate protein chains to give the final physiologically active protein.

19. Primary structure • Proteins are made up from a fixed number of amino acid residues connected to each other in a uniquelinear sequence, this is the primary structure. • The primary structure is usually depicted by a three-letter short-hand notation, e.g. ala for alanine, leu for leucine and so on arranged in the appropriate sequence.

20. Secondary Structure • The chain of amino acids that makes up the primary structure of a protein can fold itself in two ways, depending on the sequences of amino acids that are next to each other. • The alpha helix (α-helix) • The beta pleated sheet (β-pleated sheets) • Hydrogen bonds hold the folded structures in place. • This folding of the primary structure is called the secondary structure

22. Tertiary structure • This is the overall folding of the chain held by interactions between more distant amino acids. • These interactions include • Hydrogen bonds • Disulphide bridges (these are covalent bonds that form between sulphur atoms on the oxidation of two cysteine amino acids as well as ionic interactions and intermolecular forces. • Van der Waals attraction between non-polar side groups. • Ionic attractions between polar groups • Peptide bonds between molecules with more than one amine or acid group.

23. What interactions are occurring between these adjacent polypeptide chains?

25. Quaternary Structure • Separate polypeptide chains can interact together to give a more complex structure; this is known as the quaternary structure. • Haemoglobin has a quaternary structure that includes four protein chains (two α-chains and two chains) grouped together around four haem groups.

28. Contents Explain how proteins can be analysed by chromatography and electrophoresis

29. Analysis of Proteins • To determine the primary structure of a protein it must be first completely hydrolysed using the reagent concentrated hydrochloric acid and boiled for 24 hours to successively release the amino acids. • These can then be identified by: • Paper chromatography • Polyacrylamide Gel Electrophoresis PAGE.

30. Paper chromatography • Paper largely consists of cellulose fibres, these contain a large number of hydroxyl groups, making the paper quite polar. • Water molecules hydrogen-bond to these groups, so that a sheet of “dry” paper actually consists of about 10% water. • It is this water that acts as the stationary phase, the mobile phase is a solvent, either water itself or a polar organic liquid such as ethanol, propanone or ethanoic acid.

31. Activity • Carry out Paper Chromatography Expt: (?) on the mixture of amino acids to find out which amino acids are present in the mixture.

32. PolyAcrylamide Gel Electrophoresis • In PAGE the sample is placed in the centre of a polyacrylamide gel and a potential difference is applied across it. • Depending on the pH of the buffer, the different amino acids will move at different rates towards the positive and negative electrodes. • At it’s isoelectric point (the pH at which it forms a zwitterion), a particular amino acid will not move, because its charges are balanced. • When separation is complete the amino acids can be sprayed with ninhydrin and identified by comparing the distance they have travelled with standard samples or from a comparison of isoelectric points.

33. Contents B7 AHL Enzymes Describe the characteristics of biological catalysts (enzymes) Compare inorganic catalysts and biological catalysts (enzymes) Describe the relationship between substrate concentration and enzyme activity. Determine Vmax and the value of Michaelis constant (Km) by graphical means and explain its significance. Describe the mechanism of enzyme action, including enzyme substrate complex, active site and induced fit model. Compare competitive inhibition and non-competitive inhibition. State and explain the effects of heavy-metal ions, temperature changes and pH changes on enzyme activity.

34. Enzymes • Enzymes are protein molecules that catalyse biological reactions. • Each enzyme is highly specific for a particular reaction, and extremely efficient, often able to increase the rate of reaction by more than one hundred million times. • Like all catalysts, enzymes work by providing an alternative pathway for the reaction with a lower activation energy, by creating an environment in which the transition state is stabilized.

35. How enzymes work • The specificity of enzymes depends on their particular shape. • This is determined by their secondary, tertiary and quaternary structure. • The part of an enzyme that reacts with the substrate is known as the active site. • This is the part of the enzyme where the substrate (reactants) will bind and undergo chemical reaction. • The active site is not necessarily rigid, but can alter its shape to allow for a better fit – known as the induced fit theory.

36. How enzymes work • http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/animation__how_enzymes_work.html • Example: how enzymes hydrolises sucrose • http://highered.mcgraw-hill.com/sites/0072507470/student_view0/chapter25/animation__enzyme_action_and_the_hydrolysis_of_sucrose.html

37. Enzyme Kinetics • At low substrate concentrations the rate of reaction is proportional to the concentration of the substrate. • However, at higher concentrations the rate reaches a maximum, known as Vmax. • This can be explained in terms of enzyme saturation. • At low substrate concentrations there are enough active sites present for the substrate to bind to and react. • At Vmax all the sites are used up and the enzyme cannot work any faster. • Vmax was first identified by a German-born American biochemist, Leonor Michaelis (1875-1949), and a Canadian doctor, Maud Menten (1879-1960).

38. Michaelis – Menton constant Km • Michaelis and Menton went on to identify a constant in enzyme –substrate reactions. • Km, known as the Michaelis-Menton constant is the substrate concentration when the rate of the reaction is ½Vmax. • Km for a particular enzyme with a particular substrate will always be the same. • It indicates whether the enzyme functions efficiently at low substrate concentrations or whether high substrate concentrations are necessary for the reaction to be catalysed efficiently.

39. Animation: V max • http://www.wiley.com/college/boyer/0470003790/animations/enzyme_inhibition/enzyme_inhibition.htm

40. Inhibition of enzymes • Inhibitors are substances that decrease the rate of enzyme-catalysed reactions. • There are two main types of inhibitors: • Competitive inhibitors – which resemble the substrate in shape but cannot react. They slow down the reaction because they can occupy the active site on the enzyme, thus making it less accessible to the substrate. • Non-competitive inhibitors – also bind to the enzyme, but not on the active site. This is thought to cause the enzyme to change its shape so that the substrate cannot bind.

41. Animations: enzymes inhibitors • http://www.wiley.com/college/pratt/0471393878/student/animations/enzyme_inhibition/index.html • http://www.youtube.com/watch?v=PILzvT3spCQ

42. Affect of inhibitors with increasing substrate concentration • As the substrate concentration is increased, the effect of competitive inhibitors will be diminished, because there is increased competition for the active sites by the substrates. • With non – competitive inhibitors, increasing the substrate concentration will not increase the effectiveness of the enzyme, because the enzyme’s shape still remains altered by the non – competitive inhibitor.

43. Graphs to show the effect of inhibitors on Km • For competitive inhibitors Vmax is the same but Km is increased. • For non-competitive inhibitors Vmax is lower, but Km is the same.

44. Factors affecting enzyme activity • Several factors can affect the efficient functioning of enzymes. • The catalytic action of an enzyme clearly depends on its specific shape. • Its shape can be affected by the following factors • Temperature • pH • Heavy metals

45. Affect of temperature • Increasing the temperature will initially increase the rate of enzyme-catalysed reactions, because more of the reactants will possess the minimum activation energy. • The optimum temperature for most enzymes is about 40˚C. Above this temperature enzymes rapidly become denatured as the weak bonds holding together the tertiary structure become broken.

46. Affect of pH • High and low pH can also affect enzymes in a similar way as temperature. • Initially an increase in pH increases the rate of reaction until it reaches an optimum pH, the optimum pH depends on the enzyme. • At different pH values the charges on the amino acid residues change, affecting the bonds between them and so disrupting the specific tertiary structure and making the enzyme ineffective.

47. The affect of heavy metals • Heavy metals can poison enzymes by reacting irreversibly with –SH groups, replacing them with a heavy metal atom or ion so that then tertiary structure is permanently altered. • http://www.chemguide.co.uk/organicprops/aminoacids/enzymes3.html

48. Carbohydrates

49. Contents List the major functions of carbohydrates in the body Describe the structural features of monosaccharides Draw straight –chain and ring structural formulae of glucose and fructose Describe the condensation of monosaccharides to form disaccharides and polysaccharides.

50. Where do we get them from? • Carbohydrates are produced in plants by photosynthesis, this requires energy provided by sunlight. • Write the word and balanced symbol equation for photosynthesis. • When taken into the human body the process is reversed in respiration, this in turn release a lot of energy. • Write the word and balanced symbol equation for respiration. carbon dioxide + water → glucose + oxygen xCO2 + yH2O → Cx(H2O)y + xO2 glucose + oxygen → carbon dioxide + water Cx(H2O)y + xO2 → xCO2 + yH2O