Enzymes

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Unit 6

• Enzymes

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Enzymes

• Biological catalysts
• Often end in -ase
• Regulate the rate of chemical reactions in the
  cell
• Lower the activation energy needed to start the
  reaction
• Without enzymes, the reactions would depend on
  random collisions of substrates and would occur
  much more slowly.

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E S ? ES complex ? E P

• E- enzyme
• S- Substrate
• P- Product

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Enzyme and Substrate
ES complex
E S
E P
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catechol oxidaseCatechol
Oxygen-----------? Benzoquinone H2O

• Catechol-In plant cells
• When plants are damaged, the catechol and the
  enzyme catechol oxidase are exposed to air
• Catechol reacts with oxygen to form benzoquinone
• Benzoquinone has a brown color and helps protect
  damaged plants from rot
• Catalyzed by enzyme, catechol oxidase

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E S ? ES complex ? E P

• E- enzyme
• Catechol oxidase
• Source- potato
• S- Substrate
• Catechol
• Source-Lab supply
• P- Product
• Benzoquinone
• Source- produced by interaction of catechol with
  oxygen

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Enzyme and Substrate
Catechol oxidase
benzoquinone
catechol
Catechol oxidase
oxygen
ES complex
E S
E P
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(catechol oxidase) Catechol
Oxygen-------------? Benzoquinone H2O

• Todays experiment will measure the production of
  the product benzoquinone.
• We will use spectrophotometry to measure the
  absorbance of benzoquinone
• More brown pigment- more benzoquinone production

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Spectrophotometry
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Exercise 6.1

• Tube A- enzyme water
• Negative control
• Tube B- substrate water
• blank
• Tube C- enzyme substrate
• Positive control
• Reminder
• Enzyme is catechol oxidase (from potato)
• Substrate is catechol

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6.1 Procedure

• Place all 3 tubes in 40 C water bath for 10 min.
• Wavelength will be set to 540 nm
• Calibrate the spec by
• With blank in holder, set to 100 T.
• Blank will be Tube B throughout the experiment.
• Record absorbance of 3 solutions on p 65

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Which of these tubes should show benzoquinone
production?

• Tube A
• Potato extract (source of catechol oxidase)
• Distilled water
• Tube B
• 1 catechol solution
• Distilled water
• Tube C
• Potato extract
• 1 catechol solution

Negative Control- no substrate
Negative Control- no enzyme (use as blank for
all experiments)
Positive control- has substrate and enzyme
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Experiment 6.2

• Enzyme specificity
• Does catechol oxidase catalyze a reaction when a
  different substrate is used?
• Tube D
• Potato extract (catechol oxidase)
• 1 catechol (substrate)
• Tube E
• Potato extract
• 1 hydroquinone (a different substrate)

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Ex 6.2 Procedure

• 1. Set up tubes with proper solutions.
• 2. Place in 40 C water bath for 10 minutes
• 3. Record absorbance at 540 nm in Table 6.1.
• 4. Place in water bath for 10 more minutes and
  record absorbance.

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Exercise 6.3 Temperature and Enzyme Activity

• Class will be divided into 6 groups, 1 for each
  temperature.
• 1. Place 2ml of potato extract in the proper
  temperature for 5 minutes.
• 2. Add 2 ml of 1 catechol, keeping the tubes in
  the appropriate water bath.
• 3. Record absorbance after 10 minutes and after
  20 minutes.
• Note In 80 C and 100 C temperatures, a
  precipitate may form. Pipette the supernatant
  into 2 clean tubes and then take absorbance
  reading.

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• 4. Graph the absorbance readings at 20 minutes on
  Figure 6.3.
• Each group will record their data on the board.
• Which temperature do you think will be optimal
  for catechol oxidase activity?

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Exercise 6.4 Effect of pH on enzyme activity

• Each group will do the entire exercise.
• Put different pH buffers in tubes.
• Add potato extract and catechol.
• Place in 40 C water bath for 20 minutes.
• Record absorbances and graph.
• Which pH do you think will be optimal for
  catechol oxidase activity?

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Exercise 6.5 Cofactors

• Cofactor
• Some enzymes have a metal ion associated with the
  enzyme which is required for enzyme activity.
• This metallic ion is a cofactor
• Copper is the cofactor for catechol oxidase
• Will catechol oxidase catalyze the reaction if
  the copper (the cofactor) is bound by an
  inhibitor?
• EDTA will be used to bind copper and make it
  unavailable for use as a cofactor

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• Which tube would you expect to have more
  benzoquinone production?
• Tube with EDTA
• Tube without EDTA

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Exercise 6.5

• Instructor Demo
• What effect does addition of EDTA have on enzyme
  activity?