1110 Lab 3 - Metabolic Reaction Rates
Some links
Amino Acids - Wikipedia.
Amino Acids - JMol You can manipulate the amino acids.
Proteopedia This is a database of 3D protein structures.
John Kyrk's Animations lot's of nice animation on a variety of biological topics.
protein synthesis - a rather detailed animation of how ribosome synthesize proteins.
hexokinase site - This site let's you play around with the enzyme hexokinase, look at it's active site and allosteric inhibition by it's end product.
Induced Fit - nice animation of induced fit, again with hexokinase.
Induced Fit - nice animation of induced fit, this time with a cell surface receptor.
Rosetta@Home - this site let's you donate your unused computer time to figuring out how proteins fold. We have the amino acid sequence of lots of proteins we don't know the structure of.
Fold it is a program that let's you try to figure out the shape of proteins, a much more realistic version of what we do with toobers today.
Fold-it video - an example of the program above.
Protein lecture - this is a mini (4 minute) lecture on proteins and covers protein denaturation.
How to draw a graph - includes how to make a chart with Excel.
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Protein Structure
Proteins perform many roles in a cell including enzymatic and structural ones (see last lab). A protein is a chain of amino acids. We have 20 different amino acids, distinguished by their different R groups. The sequence of the amino acids determines the final 3-D structure of the protein and therefore it's function.
- primary - the actual amino acid sequence
- secondary - common shapes of the polypeptide like the alpha-helix and beta-sheets
- tertiary - specific 3 dimensional folding of the protein
- quaternary - when more than one protein forms the final complex. Dimers (2) and tetramers (4) are very common.
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| Proteins are synthesized on ribosomes. Shown here is the chemical reaction joining two amino acids via a dehydration (condensation) reaction forming a peptide bond. |
| In making our "toober protein" we used the tendency of different types of amino acids (based on their R groups) to associate with other amino acids and with their environment. |
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| Hydrophobic (water fearing) | | These are non-polar and do not form hydrogen bonds with water. They tend toward the center of a protein because of this. Long stretches may indicate a membrane protein. |
| Polar Hydrophilic (water loving) | | The R groups of these amino acids carry partial negative and positive charges (just like water does) and so can interact with water via hydrogen bonds. They therefore tend to be on the surface of a globular protein. |
| Basic and Acidic | | Basic amino acids are + charged at neutral pH while acidic are - charged. Like charges repel, opposites attract so a basic is attracted to an acidic amino acid. These charged amino acids can also interact with water. They are often involved in the active site of an enzyme. |
| Cysteine | | If this amino acid can come close to another cysteine, it can form a covalent bond(specifically a disulfide bond). Covalent bonds are stronger than the electrical attractions of polar and charged attractions. |
| Proline | | This unusual amino acid creates a kink (due to a more rigid peptide bond) in the peptide chain and so disrupts helixes and sheets. |
Catalase
Catalysts are substances that speed up chemical reactions. Enzymes are protein catalysts and control almost every reaction that takes place in a cell. Chemical reactions have one or more reactants that produce one or more products. The reactant(s) have the special name substrate in enzymatic reactions. Enzymes have a special area where the substrate(s) bind called the active site. The active site speeds up reactions in a variety of ways that, in general, reduce the energy needed to transition from the reactants to the products. They also might couple an energetically unfavorable reaction with a favorable one, such as hydrolysis of ATP to ADP. The enzyme we look at today is catalase. Most enzyme names end in -ase. Catalase catalyses the reaction 2H2O2 -> 2H20 + O2. Hydrogen peroxide (H2O2) is toxic to cells (you may well have it in your medicine cabinet) and so most cells contain catalase. We will be using catalase from potatoes. It is prepared simply by blending potatoes in water. This releases the various cellular enzymes including ones that break up proteins (called proteases). We prepare this extract every lab because of these degradative enzymes. We can measure the activity of catalase on hydrogen peroxide just by looking at the amount of foam produced by the bubbles of oxygen (one of the products - O2).
Effect of pH on reaction rate
pH is the measure of H+ concentration in a liquid and ranges from 0 to 14 with 7 being considered neutral (acidic < 7 < basic). H+ can affect both the overall shape of protein (due to changing electrical interactions between the amino acids) as well as interfering with the mechanism by which the active site works. Enzymes usually are most active at the pH of their normal environment. Most of our body fluids are at about pH 7.3 and so that tends to be an optimal pH for most of our enzymes. Pepsin is a protease secreted into our stomach, which maintains a pH of less than 2. Unsurprisingly, it's optimal activity is in that acidic range. Some bacteria and archaea live in extreme environments such as hot springs or ice and their enzymes have evolved to function optimally at those extremes (as well as extremes in pH and salt concentration).
Effect of temperature on Enzymatic activity
Temperature is a measure of kinetic energy. Most reactions occur faster at higher temperatures because molecules are moving faster and bumping into each other more often and with more energy. Enzymatic reactions usually go faster at higher temperatures until the enzyme denatures. This is because when proteins get too hot, they unwind from their precise 3D structure and no longer function.
From PSTCC's Official Practical Review
- What is an enzyme? substrate? active site? product?
- For our lab experiments, what served as the enzyme? What was the substrate? What was the product?
- How could you tell if the products (water and oxygen) had been produced?
- How did temperature affect catalase activity?
- How did pH affect catalase activity?
- Given a series of tubes, be able to identify the tubes with the highest catalase activity and the least catalase activity.
- What does it mean to denature an enzyme?
- Why is enzyme shape important to enzyme function?
- What determines the shape of an enzyme or other protein? How can heat alter the shape?? How can pH changes alter shape? Why?
- How were the toober and pushpins used to model protein structure?
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Copyright 2010 by Douglas Dodd, Ph.D., all rights reserved.