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9.7 The Biochemistry of Movement: 4. Proteins

Syllabus reference (October 2002 version)
4. Proteins are used as both structural molecules and as enzymes to catalyse metabolic reactions	Students learn to: describe the composition and general formula for amino acids identify the major functional groups in an amino acid outline the nature of a peptide bond and, using a specific example, describe the chemistry involved in the formation of a peptide bond explain, using a named example, the relationship between the chemical features of a protein and its shape using appropriate diagrams or models account for the shape of a protein molecule in terms of electrostatic forces hydrogen bonding forces hydrophobic forces disulfide bonds account for the process of protein denaturation identify enzymes as a special class of proteins with a binding site that is substrate specific using a named example of an enzyme, explain why the enzyme’s binding site is substrate specific	Students: process information from secondary sources to draw the generalised structural formula for an amino acid identify data, plan, choose equipment and perform first-hand investigations to observe the effect of changes in pH and temperature on the reaction of a named enzyme and use the available evidence to relate this to possible changes in the primary, secondary and/or tertiary structure of the enzyme involved process and analyse information from secondary sources to discuss the use of models in the development of understanding of enzyme function

Extract from Chemistry Stage 6 Syllabus (Amended October 2002) © Board of Studies, NSW.
[Edit: 7 Jul 09]

Background: Proteins are large molecules (macromolecules) made in our cells from building blocks called amino acids. Amino acids are obtained from our food. Thousands of different proteins are made, some to be enzymes, some to form structural parts of our body such as hair, nail, tendons, ligaments and some, called antibodies, to act against foreign substances entering the body.

process information from secondary sources to draw the generalised structural formula for an amino acid

describe the composition and general formula for amino acids identify the major functional groups in an amino acid

• Identify the amino group –NH₂ and the organic acid group –COOH (carboxyl group)
  
  
• Process information on the structure of amino acids, drawing or observing several to generalise the common components as NH₂-CH-COOH. Circle this amino acid part in a diagram or model and identify that it consists of both an amino functional group and a carboxyl functional group joined through the same C atom. Relate the composition to the name.
  
  
• By processing information such as structural diagrams or models of amino acids identify the side chain part of the molecule, recognising that there are 20 different side chains and classifying them as hydrophobic (hydrocarbon only), containing a second amino group or acid group, containing an OH able to H-bond or containing sulfur. It is not necessary to learn the structures of the side chains.
  
  
• Using the symbol R for the various side chains describe the general formula of an amino acid as

  

• From the generalized structural formula note that an amino acid is composed of hydrogen, carbon, nitrogen and oxygen. Except for two amino acids which contain sulfur, H, C, N and O are also the only elements found in the side chains R.
  
  
• Recall that the –COOH group is a weak acid and can dissociate to form –COO^- and H⁺ in water.

  -COOH -COO^- + H⁺

• Given that the –NH₂ can act as a weak base, the following equilibrium also exists in water

  -NH₂ + H⁺ -NH₃⁺

• That is, amino acids are both weak acids and weak bases and at neutral pHs, the functional groups are usually

  

  The –COOH group has donated its proton to the NH₂ group.

• Side chains containing an amino or carboxyl group will also be ionised at neutral pHs and therefore able to form ionic bonds because of their + or – charge.

  

outline the nature of a peptide bond and, using a specific example, describe the chemistry involved in the formation of a peptide bond

• Amino acids can join together in long polymer chains called polypeptide chains. The functional groups are responsible for the joining of the amino acids, the amino group of one amino acid joining to the carboxyl group of another. Having these groups exist as a positive –NH₃⁺ and a negative –COO^- is important in amino acids attracting one another to form polypeptide chains.
  
  
• The bonds joining amino acids are called peptide bonds. A water molecule is lost as the peptide bond forms. Describe peptide bond formation using structural formulas.
  
• Amino acids keep joining on by forming more peptide bonds. Polypeptide chains vary in length from 50 to 2000 amino acids. When formed, the polypeptide chain folds over to form the final 3-D shape. A folded polypeptide chain is called a protein.
  
  
• Outline how hydrogen bonding involving the O of one peptide linkage and the H attached to a N of another peptide linkage can be important in attracting and stabilizing different parts of a polypeptide chain:

  

• The exact sequence of the amino acids in a chain is important as this determines the way the chain will fold and this, in turn, gives the protein its final shape and function.
  
  
• Genes determine the sequence of the amino acids in a polypeptide chain and so genes control the synthesis of the body’s proteins. Many proteins provide structural frameworks in cells and tissues while others, called enzymes, catalyse metabolic reactions.

explain, using a named example, the relationship between the chemical features of a protein and its shape using appropriate diagrams or models

account for the shape of a protein molecule in terms of

1. electrostatic forces
2. hydrogen bonding forces
3. hydrophobic forces
4. disulfide bonds

• Explain how once a polypeptide chain is formed, the side chains account for how it will fold.
  
  The side chains can form bonds with other side chains:
• side chains that contain an ionic group can form electrostatic (ionic) bonds with oppositely charged ionic groups on other side chains.
  
• side chains with O or N atoms can H-bond to other side chains. H-bonds also form between C=O and N-H groups in the peptide links.
  
• Non-polar hydrocarbon side chains attract through dispersion forces. These attractions often form spiral helices which exclude water.
  
• the amino acid with an –SH group on its side chain, called cysteine, can join up with another cysteine side group on another part of the chain. The two cysteine side groups will react to form a strong covalent bond called a disulfide bond (-S-S-).
  
• The way in which the side chains cause the folding can be modelled. Long pieces of flexible plastic (eg blue packing strip plastic) can represent the primary chain structure (amino acid sequence). Attach different objects to represent different types of side chains eg. pieces of straws with sticky tape at the ends to represent side groups with –SH. Bring the appropriate side groups together into a 2-Dimensional shape, including some helical areas with clusters of hydrophobic side groups. Identify this as secondary structure. Further 3-Dimensional folding is tertiary structure.
  
  Protein structure Access Excellence @ the national health museum, USA.
• Seek out a model or diagram of a short polypeptide chain such as the hormone insulin, showing the amino acid sequence (primary structure) and conformation (folding into secondary and tertiary structure). The illustration should show the positions of cysteine side chains and hence the position of –S-S- bonds holding the secondary and tertiary shape together.
  
  
• Explain that the positions of the cysteine side groups are important in determining the shape. If the cysteine had been in different positions, the disulfide bonds would cause a different shape to occur. The same principle accounts for shape being determined by the position of other side chain bonding groups, eg. ionic groups.

Insulin structure showing all atoms except hydrogen Washington University, USA.
Levels of protein structure exemplified by Insulin Biotopic, UK.

identify data, plan, choose equipment. and perform first-hand investigations to observe the effect of changes in pH and temperature on the reaction of a named enzyme and use the available evidence to relate this to possible changes in the primary, secondary and/or tertiary structure of the enzyme involved

• Using a given enzyme reaction plan first-hand investigations to observe the effect of temperature and pH on the enzyme activity. Design the first-hand experiments and choose suitable equipment with guidance from your teacher.Variables to be controlled are quantities of substrate and enzyme used. In one experiment, only temperature is varied and in a second experiment, only pH is varied. Appropriate controls with water instead of enzyme should be set up. The plan should include a risk assessment.
  
  
• Suggested reactions are liver or potato catalase with hydrogen peroxide substrate or rennin in junket tablets with milk as substrate. Amylase and starch or protease and gelatin are also possibilities. If catalase is used, the liver or potato should be ground in a mortar and if junket tablets are used they should also be crushed. The amount of enzyme used should be much smaller than the amount of substrate. With catalase, the amount of frothing from the oxygen gas released can be used as the measure of activity; with rennin, the time taken for the milk proteins structure to change and the milk to clot. Enzyme preparation and substrate samples should both be brought to the same temperature before mixing and observing activity. With pH, enzyme can be mixed with chosen buffered pH solutions and then added to substrate, all samples at room temperature.
  
  
• The identified data (results) should be discussed, relating the changes in activity to changes in the folding of the amino acid chain of the enzyme. As pH changes, ionic bonds and H-bonds are disrupted. As temperature increases from 0⁰C, the rate of reaction is increased up to a maximum activity after which activity is suddenly lost as the heat causes the movement of the chain to increase to a point where bonds are broken and shape is lost.

account for the process of protein denaturation

• When the folding of the polypeptide chain of a protein is changed, the shape is lost and the protein function is lost.
  
  
• Any change in the shape which causes loss of function is called denaturation.
  
  
• Account for the loss of the shape as caused by disruption of the bonds between the side chains such as breaking of ionic bonds, H-bonds, disulfide bonds. For example lowering the pH causes –COO^- groups to become uncharged –COOH groups and this destroys ionic bonds.

Denaturation of a protein structure Virtual Chembook, Elmhurst College, Chicago, Illinois, USA .

process and analyse information from secondary sources to discuss the use of models in the development of understanding of enzyme function

identify enzymes as a special class of proteins with a binding site that is substrate specific

using a named example of an enzyme, explain why the enzyme’s binding site is substrate specific

• Process and analyse information on the early model of enzyme activity called the lock and key model.
  
  
• Use plasticine, playdough or other flexible material to model. Make a shape representing an enzyme with a particular pattern at one spot as the active site. Only substrates which fit this site will react. Model an enzyme reaction with substrate binding, being altered and then being released, the enzyme catalyst being unchanged.
  
  
• Discuss the observations made possible by use of the lock and key model – Substrate is altered, enzyme unchanged, one enzyme molecule can transform many substrate molecules.
  
  
• Identify that substrates must be able to fit the active site in order to undergo a reaction ie. the enzyme is specific. Anything that causes the loss of shape of an enzyme will interfere in the reaction with the substrate. Binding of the enzyme to the substrate uses intermolecular forces.
  
  
• The lock and key model provided a 3-D picture of the way enzymes work. Using this model explain why catalase only decomposes a specific substrate, hydrogen peroxide.
  
  
• Summarise that enzymes are proteins that catalyse reactions in living things. They have a particular area called an active site that can bind the substrate by means of intermolecular forces. Only substrates that match or fit the active site will react. These substrates must be able to form bonds with the active site. Therefore enzymes are substrate specific.
  
  
• An example of the fit between enzyme and substrate is seen with the digestive, protease enzyme called chymotrypsin which hydrolyses certain peptide bonds in proteins in our diet. As part of its active site, this enzyme has a deep channel which binds the bulky hydrophobic side chains of the substrate protein. This brings the peptide bonds near hydrophobic side chains into a position where they are cleaved. This explains why chymotrypsin breaks specific peptide bonds which are next to large hydrophobic side.

Lock and key enzyme model , Florida State University, Florida, USA.