Unit 6 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.

E + S  ES complex  E + P E- enzyme S- Substrate P- Product

Enzyme and Substrate E + S ES complex E + P

catechol oxidase Catechol + Oxygen  Benzoquinone + H 2 O 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

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

Enzyme and Substrate E + S ES complex E + P Catechol oxidase catechol Catechol oxidase benzoquinone oxygen

(catechol oxidase) Catechol + Oxygen  Benzoquinone + H 2 O Today’s 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

Spectrophotometry

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

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

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

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)

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 Place in water bath for 10 more minutes and record absorbance.

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.

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?

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?

Exercise 6.5 Cofactors Cofactor required –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

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

Exercise 6.5 Instructor Demo What effect does addition of EDTA have on enzyme activity?