PRACTICAL LAB 2

ENZYMES

OBJECTIVES

  • Objective 1: Understand the concept of optimal conditions and its relation to enzymatic activity.

  • Objective 2: Comprehend the mechanisms by which temperature, pH, inhibitors, and substrate concentration influence the rate of enzymatic reactions.

PRIOR READING FOR PRACTICAL LAB 2

  • Review Concept 6.4 (pp. 135-140) from the textbook.

INTRODUCTION

  • Thousands of organized chemical reactions occur in a cell every minute.

  • Reactions are regulated by biological catalysts known as enzymes, which accelerate chemical processes.

  • Example of spontaneity: Hydrogen and oxygen gases react to form water but do so at a negligible rate without a catalyst.

  • Example of temperature influence: Wood reacts gradually with oxygen, but combustion requires significant heat.

  • Enzymes are essential in cells to facilitate reactions at low temperatures where heat would be damaging.

ENZYME FUNCTIONS

  • Definition of Enzymes:

    • Biological catalysts that speed up chemical reactions.

  • Characteristics of Enzymes:

    1. Enzymes are composed of proteins.

    2. Each enzyme selectively reacts with a specific substrate (reactant).

    3. They do not require high heat to induce chemical reactions.

    4. Enzymes do not change the equilibrium concentrations of a reaction but accelerate the process.

    5. They do not impact the free energy changes ($\Delta G$) of a reaction.

    6. Enzymes remain unaltered post-reaction and can catalyze subsequent reactions.

ENZYME-SUBSTRATE COMPLEX

  • Formation of the enzyme-substrate complex:

    • Enzymes and substrates interact at the active site, forming the enzyme-substrate complex.

    • The general reaction can be summarized as:

    • E+S↔ES↔E+PE + S \leftrightarrow ES \leftrightarrow E + P

    • The formation of this complex allows the substrate's chemical bonds to be rearranged, resulting in product formation.

FACTORS AFFECTING ENZYME ACTIVITY

  • Enzyme activity is affected by environmental factors:

    • pH and temperature modifications can alter the enzyme's three-dimensional shape and consequently its activity.

    • Inhibitors impact enzyme function; they can be classified into:

    • Activators: Chemicals essential for enzyme activity.

    • Cofactors: Non-protein substances vital for enzyme activity, which can be organic (coenzymes) or inorganic (metal ions).

    • Inhibitors: Compounds that reduce enzyme activity, categorized into:

      • Competitive Inhibitors: These resemble substrates and compete for the active site.

      • Non-competitive Inhibitors: These bind to an alternative site on the enzyme, altering its function without preventing substrate access.

  • Methods of measuring enzyme activity:

    1. Rate of substrate disappearance.

    2. Rate of product appearance.

  • Focus for the lab:

    • Investigate the activity of catechol oxidase, its inhibitors, and the effect of temperature and pH on its function.

CATECHOL OXIDASE

  • Occurs in potatoes and catalyzes the oxidation of catechol to benzoquinone in the presence of oxygen, producing a pigment and darkening fruits/vegetables.

EXERCISE I: ACTION OF CATECHOL OXIDASE

  • Objective: Test for catechol oxidase presence and observe reaction products.

  • Safety Note: CATECHOL IS A POISON!

    • Follow the Safety Data Sheet (SDS) guidelines.

  • Procedure Overview:

    1. Turn on the spectrophotometer; set at 500 nm and 0-1 absorbance.

    2. Prepare three test tubes with specified solutions according to Table 1, ensuring equal total solution volumes.

TABLE 1: CONTENTS OF TEST TUBES FOR EXERCISE I

  • | TUBE | DISTILLED WATER | CATECHOL | DISTILLED WATER | POTATO EXTRACT |

    • |------|-----------------|-----------|--------------|-----------------|

    • | 1 | 5 ml | 10 drops | 10 drops | --- |

    • | 2 | 5 ml | 10 drops | --- | 10 drops |

    • | 3 | 5 ml | --- | 10 drops | 10 drops |

EXERCISE II: INHIBITING THE ACTION OF CATECHOL OXIDASE

  • Objective: Investigate inhibition by phenythiocarbamide (PTC).

  • Inhibition Types:

    • Competitive Inhibition: Inhibitor competes with substrate for active site; can be overcome by increasing substrate concentration.

    • Non-competitive Inhibition: Inhibitor binds to a site other than the active site, permanently altering enzyme function; increasing substrate concentration does not reverse inhibition.

  • Procedure Overview:

    1. Prepare three test tubes as per Table 3, ensuring sequential addition of reagents.

TABLE 3: CONTENTS OF TEST TUBES FOR EXERCISE II

  • | TUBE | DISTILLED WATER | POTATO EXTRACT | PTC | DISTILLED WATER | CATECHOL |

    • |------|-----------------|----------------|-----|-----------------|-----------|

    • | 1 | 5 ml | 10 drops | 10 drops | 10 drops | 10 drops |

    • | 2 | 5 ml | 10 drops | 10 drops | --- | 20 drops |

    • | 3 | 5 ml | 10 drops | --- | 20 drops | 10 drops |

EXERCISE III: THE EFFECT OF pH ON CATECHOL OXIDASE ACTIVITY

  • Objective: Determine the optimum pH for catechol oxidase activity.

  • Note on procedure: High acidity can precipitate proteins, impeding accuracy.

  • Procedure Overview:

    1. Prepare five test tubes according to Table 5 with specified solutions.

TABLE 5: CONTENTS OF TEST TUBES FOR EXERCISE III

  • | TUBE | DISTILLED WATER | POTATO EXTRACT | NaOH | DISTILLED WATER | CATECHOL |

    • |------|-----------------|----------------|------|-----------------|-----------|

    • | 1 | 5 ml | 10 drops | --- | (pH 6) | 10 drops |

    • | 2 | 5 ml | 10 drops | 1 drop | (pH 8) | 10 drops |

    • | 3 | 5 ml | 10 drops | 2 drops| (pH 8.5) | 10 drops |

    • | 4 | 5 ml | 10 drops | 3 drops| (pH 9.0) | 10 drops |

    • | 5 | 5 ml | 10 drops | 4 drops| (pH 9.5) | 10 drops |

EXERCISE IV: THE EFFECT OF TEMPERATURE ON CATECHOL OXIDASE ACTIVITY

  • Influence of Temperature: Chemical reactions generally increase with temperature. Each $10^{\circ}C$ rise can double or triple reaction rates, but extreme heat may denature proteins.

  • Procedure Outline:

    1. Design an experiment to test catechol oxidase activity across five temperature environments: 0°C, room temperature, 40°C, 60°C, and 80°C.

QUESTIONS TO CONSIDER

  1. Graphical representation of results.

  2. Explanation of anticipated results.

RESULTS RECORDING TABLES

  • Record absorbance values for each exercise as indicated in Table 2, Table 4, and Table 6.