ENZYME QUESTION - 1968 L. PETERSON/AP BIOLOGY Suppose that you have isolated an extract from a tissue and you have found that the extract speeds up the rate of a particular reaction. What kind of information would you need to demonstrate that the substance responsible for increasing the rate of this reaction is an enzyme? Explain how this information would indicate that the catalytic effect is due to an enzyme. STANDARDS: maxiumum points = 13 PROOF THAT IT IS ORGANIC (1/2 PT) PROOF THAT IT IS PROTEIN (1 PT) PROTEIN EVIDENCE: (1 PT EACH/MAX, = 5) DENATURED under different pH or temperatures MACROMOLECULE will diffuse through DIALYSIS TUBING if acted on by protease centrifugation - large molecule - will be in precipitate NINHYDRIN will turn blue in presence of amino acids CHROMATOGRAPHY RATE OF REACTION (1/2 PT) (2 PTS EACH/MAX. = 4) vary amount of substrate - determine changing rate of reaction; should level-off once maximum turnover rate is reached; vary pH & temperature - rate of reaction will be at maximum & drop off radically on either side; INTERACTION BETWEEN ENZYME & SUBSTRATE (1/2 PT) X-ray Diffraction proof of changes in shape of "enzyme" (3/2 PTS) ENZYME QUESTION - 1969 L. PETERSON/AP BIOLOGY Proteins functioning as enzymes exhibit precise specifications. Discuss the levels of structural organization within proteins which are responsible for specific molecular interactions. STANDARDS: maximum points = 20 PRIMARY STRUCTURE (1/2 PT) (2) amino acid sequence & number / determining other structures peptide bonds SECONDARY STRUCTURE (1/2 PT) folding within polypeptide chain (4) H-bonds Disulfide bonds Alpha Helix - globular proteins Beta Configuration - fibrous proteins TERTIARY STRUCTURE (1/2 PT) further folding of alpha helix (4) H-bonds Disulfide bonds electrostatic forces (interactions) van der Waals forces QUATERNARY STRUCTURE (1/2 PT) (2) Conjugated Proteins - many polypeptide chains ENZYMES ARE SPECIFIC BECAUSE OF ACTIVE SITE (6) particular shape of molecule particular charge distribution Coenzymes or cofactors may be required Mention of Lock-Key Hypothesis Mention of Induced-Fit Hypothesis EXAMPLE ENZYME QUESTION - 1985 L. PETERSON/AP BIOLOGY Describe the chemical compositions and configuration of enzymes and discuss the factors that modify enzyme structure and/or function. STANDARDS: maximum points = 20 CHEMICAL COMPOSITION AND CONFIGURATION Proteins/Large Molecules/ Polypeptides/CHONS/CN Terminals (2 max) Prosthetic Groups/Metal Atoms/Apoenzyme/Coenzyme/Cofactor (2 max) Primary and Secondary with Discussion (2 max) Tertiary OR Quaternary with Discussion / Globular (2 max) Specificity or "Lock and Key" (1 pt) Peptide Bonds/Covalent Bonds for Primary Folding to Form a Groove, Active Site (1 pt) 3D Configuration due to Van der Waal, R group, ionic, etc. (1 pt) Hydrophobic/Hydrophilic (1 pt) Maximum = 8 FACTORS THAT MODIFY ENZYME ACTION AND/OR FUNCTION: Temperature = 1 Discussion of Effect on Structure = 1 Denatures Protein/3D shape altered = 1 (reaction rate changes) pH = 1 Discussion of Effect on Structure = 1 Breakage of weak bonds changes enzyme's shape = 1 Discussion of Effect on Function = 1 (altering of active site results in inability of substrate to bond at site) Allosteric Inhibition Discussion of Effect on Structure = 1 = 1 (binding of an effector on an enzymes changes shape of enzyme) Discussion of Effect on Function = 1 (results in activation or inactivation) Competitive Inhibition Discussion of Effect on Function = 1 = 1 (competitive molecule binds to active site blocking it/NO rx) May reverse action by increasing substrate/enzyme concentration = 1 Irreversible or Noncompetitive Inhibition = 1 Discussion of Effect on Function (binding of molecule blocks functional groups at active site) = 1 Activation of an Enzymatic Precursor on structure = 1 (pepsinogen to pepsin) Effect on Functions = 1 (nonfunctional to active) Genetic mistakes Modify Structure/Function of Enzymes = 1 Feedback Inhibition relative to control = 1 Induced Fit - binding of substrate to enzyme alters shape = 1 Amount of substrate to Amount of enzyme - modifies function = 1 Reversible/Irreversible denaturation = 1 Factors may change energy of activation = 1 Ionic Factors affect structure and/or function = 1 Binding of Effector to speed activation/deactivation = 1 Maximum = 12 ENZYME QUESTION - 1988 L. PETERSON/AP BIOLOGY 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 (sec) 0 10 20 30 40 50 60 Product formed (mg) 0.00 0.25 0.50 0.70 0.80 0.85 0.85 Draw a graph of these data and answer the following questions. a. What is the initial rate of this enzymatic reaction? b. What is the rate after 50 seconds? Why is it different from the initial rate? 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? d. How might altering the substrate concentration affect the rate of the reaction? Why? e. How might altering the pH affect the rate of reaction? Why? STANDARDS: maximum points = 10 DATA RECORD AND CALCULATIONS GRAPH axis X = Time (ind); Y = Product (dep) 3 pts scale and label axis curve plotted - drawn curve necessary a. initial rate 1 pt. setup (.25-.00)/(10-0) 2 pts 0.025 mg/sec or (.50-.00)/(20-0) .25 mg/10 sec or number 0.025 1.5 mg/min 1 pt. units (mg/sec) or mg/min) 1/40 mg/sec b. rate after 50 sec 1 pt Zero 1 pt. set up (.85-.85)/(60-50) or 1 pt. units if not awarded in part a. net rate equilibrium Why? 1 pt Substrate gone or reaction at equilibrium Other explanation - any are possible Product inhibition Product changes pH or temp optimum Product release time varies Maximum = 7 pts EXPLANATIONS: c. Temperature variation Change: stops reaction; no product formation; 1 pt rate near or at zero Explanation: Conformational shape change - denaturation 1 pt (inactivation - "kills" in quotes) d. Substrate concentration variation; Change: 1 pt (Increase) a) no change, initial slope same; longer to level off; or b) increase in reaction rate and/or (Decrease) more gentle slope; decrease rate or take less time to level off; Explanation: 1 pt (Increase) a) Enzyme is working as fast as it can (Vmax) or b) It will approach Vmax or (Decrease) Enzyme no longer saturated; or further from saturation; e. pH variation Change: 1 pt a) Slight change may affect the curve either way b) Drastic change may stop the reaction Explanation: 1 pt a) Enzyme has optimum pH b) Enzyme can be denatured by extremes Maximum = 6 pts ENZYME QUESTION - 1994 L. PETERSON/AP BIOLOGY Enzymes are biological catalysts. a. Relate the chemical structure of an enzyme to its specificity and catalytic activity. b. Design a quantitative experiment to investigate the influence of pH or temperature on the activity of an enzyme. c. Describe what information concerning the structure of an enzyme could be inferred from your experiment. Since the question asked students to respond with both specific facts about enzymes and broad conceptual statements about the design of an experiment, these standards reflect both approaches. In understanding how an enzyme could be affected by a quantitative experiment with temperature or pH, students not only had to state specific features such as the three dimensional shape of an enzyme, but they also had to describe how to control variables in an experiment. Finally, students were expected to apply the results of their experiment to changes in the structure of the enzyme. Structure and catalytic activity of enzyme (maximum of 4 points) __ protein or amino acids (and/or others, such as ribozyme) __ 3-D shape/levels of structure (primary, secondary, teritary, etc.) __ bonding explanation of structure (alpha helix, hydrophobic interactions, van der Waals forces, etc.) __ active site ("groove", "pocket") / special shape for substrate / "lock and key" __ modifiers of enzyme shape (cofactors, activators, inhibitors) __ induced fit theory (function of enzyme - substrate fit) __ activation energy lowered __ substrate altered Experimental design (maximum of 5 points) Experiment based on enzymatic activity / inital choice of temperature or pH is binding __ eliminate other variables (conc., amounts, time, pH, temp in alternate experiment) __ negative control (setup without enzyme or without substrate) __ describe experimental variable (temperature or pH) values or range __ uses correct enzyme-substrate pair __ measure disappearance of substrate, 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 (exemplary set-up; indep, dep variables identified; rate calculation or explanation; replication of experiment, etc.) Inference from experimental design (maximum of 2 points) __ correct link of predicted results to changes in enzyme structure a. range of activity implies slight change in shape OR b. loss of activity implies denaturation OR c. no loss in activity implies no change in structure __ elaboration on changes in enzyme structure (conformation explanation, bonding shifts or an explanation of why no change in activity is predicted)