The activation energy is the difference between the energy of the reactants and the tip of the “hump” on the diagram above. RATE = change in a measurable quantity Change in time How Enzymes Work Would this reaction be endergonic or exergonic? Macromolecules & the Enzymes that Catalyze the Reactions Polymerase —catalyzes polymer formation using dehydration synthesis Hydrolase— catalyzes the breakdown of polymers using hydrolysis reactions Why do we store hydrogen peroxide in a brown bottle? Remember enzymes are proteins with primary, secondary, tertiary and quaternary structure. Proteins usually end in the suffix –in (insulin, actin). If the protein is an enzyme, it often ends in –ase (lactase, catalase) Can’t fit! “conformation” = shape! The shape of the enzyme and more specifically, it’s active site determines its function. All these H+ ions made an acidic environment that denatured the protein we call an enzyme. The active site is rendered useless. When you see a “bell curve” or normal distribution, you’re dealing With an optimum. Drop a perpendicular Line down from the peak of the “bell” and Read the x-axis to determine The optimum. BOTH temperature AND pH exhibit This type of a result in most Biological settings. It’s the 3 Bears scenario: Too much, Too little, and JUST RIGHT! Feedback Inhibition:The product forms shuts down the process by binding the enzyme at an allosteric site! Yeah, I hate them, too! This is a REDOX titration. “OILRIG” “Oxidation is loss” of electrons “Reduction is gain” of electrons. These two processes are always happening together. We call them a set of “coupled” reactions. YOU HAVEN’T HEARD THE LAST OF REDOX! If you get the “purple stuff” on your skin, It will stain! H2O2 reduces KMnO4 on your skin to the colorless form And thus, removes the Stain! Use it on the buret As well. Fancy word for portion. Allows for RELATIVE data. This reduces error. It’s not important that you get the same color as your neighbor—it’s important you agree with YOURSELF on each titration. Much easier to read If your buret has WHITE marks! I call this an easy question. You’ll call it a TRICK question! Multiple Choice Question: E1 E2 E3 W X Y Z 1. In the series of enzyme reactions shown above, product Z is able to occupy the active site of enzyme E2. Product Z can therefore first inhibit the production of (A) W (B) E1 (C) X (D) E2 (E) Y Multiple Choice Question: 2. A substrate molecule may be bound to the active site of an enzyme by all of the following EXCEPT (A) hydrogen bonds (B) peptide bonds (C) ionic bonds (D) van der Waals interactions (E) hydrophobic interactions For the next 3 questions: Multiple Choice Question: 3. In an experiment to test the effect of amylase on starch, the control would be (A) flask A only (B) flask B only (C) flask C only (D) flasks A and B (E) flasks A and C Multiple Choice Question: 4. After 2 minutes, a positive test for sugar would most likely be observed in (A) flask A only (B) flask B only (C) flask C only (D) flask A and C (E) flask B and C Multiple Choice Question: 5. Support for the hypothesis of enzyme denaturation can be obtained by comparing starch digestion in (A) flasks A and B after 5 minutes (B) flasks B and C after 5 minutes (C) flasks A and C after 5 minutes (D) flask A at time zero and again after 5 minutes (E) flask B at time zero and again after 5 minutes Multiple Choice Question: Questions 6 - 8 In a laboratory experiment using spectrophotometry, an enzyme is combined with its substrate at time zero. The absorbance of the resulting solution is measured at time zero and at five-minute intervals. In this procedure an increase in absorbance is related to the amount of product formed during the reaction. The experiment is conducted using the three preparations shown in the table below. Absorbance Enzyme Preparation 0 min 5 min 10 min 15 min 20 min I. 3 mL of enzyme preparation 0.0 .22 .33 .38 .37 3 mL of substrate pH 5.0 II. 3 mL of boiled enzyme preparation 0.0 .06 .04 .03 .04 2 mL of substrate pH 5.0 III.3 mL of enzyme preparation 0.0 .32 .37 .36 .38 2 mL of substrate pH 6.0 Multiple Choice Question: 6. The most likely reason for the failure of the absorbance to increase significantly after 10 minutes in preparation III is that (A) the reaction is thermodynamically impossible at pH 6.0 (B) the enzyme is not active at this pH (C) a pH of 6.0 prevents color development beyond an absorbance of .38 (D) the enzyme is degraded more rapidly at pH 6.0 than it is at pH 5.0 (E) most of the substrate was digested during the first 10 minutes Multiple Choice Question: 7. Which of the following statements is best supported by the data? (A) Increasing the pH to 7.0 would yield an absorbance higher than 0.30 after 5 minutes. (B) The enzyme demonstrates more activity at pH 6.0 than at pH 5.0. (C) The enzyme has no activity at pH 6.0. (D) A pH of 5.0 is the optimum for the activity of the enzyme. (E) The enzymatic activity is independent of pH. Multiple Choice Question: 8. Which of the following can best be concluded from a comparison of the results of preparation II with those of preparation I? (A) Heating the enzyme is not required to increase the absorbance. (B) Boiling does not break down the substrate. (C) Most of the increase in the amount of product in preparation I was due to an enzymatic degradation of the substrate. (D) Enzymatic reactions proceed at a faster rate after boiling the enzyme. (E) Products resulting from the breakdown of the enzyme are responsible for the absorbance increase in preparation II. Multiple Choice Question: 9. Which of the following can be used to determine the rate of enzyme-catalyzed reactions? (A) Rate of disappearance of the enzyme (B) Rate of disappearance of the substrate (C) Rate of disappearance of the product (D) Change in volume of the solution (E) Increase of activation energy Multiple Choice Question: 10. Within the cell, many chemical reactions that, by themselves, require energy input (have a positive freeenergy change) can occur because the reactions (A) may be coupled to the hydrolysis of ATP (B) take place very slowly (C) take place when the cells are at unusually high temperatures (D) are catalyzed by enzymes (E) are aided by various metal ions that act as catalysts For the next 4 questions: Refer to the following graph. The solid curve and the dashed curve represent alternate pathways for the same reaction. One pathway is enzyme catalyzed. Multiple Choice Question: 11. Represent the activation energy of the enzymecatalyzed reaction Multiple Choice Question: 12. Represents the net energy change of the reaction Multiple Choice Question: 13. Represent the energy state of the products of the enzyme-catalyzed pathway Multiple Choice Question: 14. Represents the energy state of the products of the pathway that is not enzyme-catalyzed FR Questions… 1994: Enzymes are biological catalysts. a. Relate the chemical structure of an enzyme to its specificity and catalytic activity. FR Questions… 1994: a. 4 points maximum Enzymes are biological catalysts. Relate the chemical structure of an enzyme to its specificity and catalytic activity. •Protein with details of structure •Primary, secondary, tertiary, quaternary structure explained and related to 3-D shape •Bonding [α- helix, β-pleated sheet, H-bonding, S-S bridges, hydrophobic interactions •Active site—lock and key—substrate •Activators, inhibitors •Induced fit theory •Activation energy lowered •Substrate altered FR Questions… 1994: Enzymes are biological catalysts. b. Design a quantitative experiment to investigate the influence of pH or temperature on the activity of an enzyme. FR Questions… 5 points maximum 1994: Enzymes are biological catalysts. b. Design a quantitative experiment to investigate the influence of pH or temperature on the activity of an enzyme. •Eliminate, control other variables •Negative control [setup without enzyme OR enzyme without substrate] •Describe experimental variable [temp or pH] VALUES or RANGES •Use correct enzyme—substrate pair! •Measure disappearance of substrate or appearance of product, heat production, etc. •Report data, predicted results, such as loss of activity, reduced activity, or no change in activity •Elaboration of experiment—rate calculation explained, identify independent and dependent variables, repeat for reliability, etc. FR Questions… 1994: Enzymes are biological catalysts. c. Describe what information concerning the structure of an enzyme could be inferred from your experiment. FR Questions… 2 points maximum 1994: Enzymes are biological catalysts. c. Describe what information concerning the structure of an enzyme could be inferred from your experiment. •Correct link of predicted results to changes in enzyme structure •Range of activity implies slight change in enzyme STRUCTURE •Loss of activity implies denaturation [must explain denaturation] •No loss in activity implies no change in structure •Elaboration on changes in enzyme structure •Conformation explanation, bonding shifts •Explanation of why no change in activity is predicted FR Questions… 1988: After an enzyme is mixed with its substrate, the amount of product formed is determined at 10-second intervals for 1 minute. Data from this experiment are shown below: Time (s) Product Formed (mg) 0 10 0.0 0.25 20 30 40 50 60 0.50 0.70 0.80 0.85 0.85 Draw a graph of these data and answer the following questions. Graph of the data: Of course, your graph would be hand sketched! 3 points! •Time on x (independent) & product on y-axis (dependent) •Scale and Label axes •Plot points— Curve MUST Be drawn To earn this Point! RATE = change in a measurable quantity = (0.25-0.0 mg) = 0.025 mg/s INITIAL RATE Change in time (10-0 sec) 3 points! FR Questions… a. What is the initial rate of this enzymatic reaction? INITIAL RATE = change in a measurable quantity = (0.25-0.0 mg) = 0.025 mg/s Change in time (10-0 sec) b. What is the rate after 50 seconds? Why is it different from the initial rate? RATE after 50 sec = change in a measurable quantity = (0.85-0.85 mg) = 0.0 mg/s Change in time (60-50 sec) since EQUILIBRIUM has been achieved [the graph flattens out or levels off!] •Setup for Rate calculation •Units correct •Why? Equilibrium Has been reached! FR Questions… c. What would be the effect on product formation if the enzyme were heated to a temperature of 100 oC for 10 minutes before repeating the experiment? Why? Stops reaction; no product formation; rate near or at zero 1 pt Conformational shape change - denaturation 1 pt FR Questions… d. How might altering the substrate concentration affect the rate of the reaction? Why? Change: (1 pt) •Increase amt. of substrate—no change in rate, initial slope the same! May take longer to level off. •Decrease amt. of substrate— decrease the slope, decrease the rate—takes less time to level off. Explanation: (1pt) •Increase—Enzyme becomes saturated and is working as fast as it can! •Decrease—Enzyme is no longer saturated or at least further from saturation FR Questions… e. How might altering the pH affect the rate of reaction? Why? Change:(1pt) Slight change may affect the curve either way Drastic change may stop the reaction Explanation:(1pt) Enzyme has an optimum pH Enzyme can be denatured by extremes