Tuesday, July 12, 2011

Enzymes


            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)


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