ENZYME CATALASE

Introduction: Enzymes are proteins one of the four macromolecules: carbohydrates, proteins, lipids, and nucleic acids. They are produced by cells to act as a catalyst in chemical reactions. Enzymes are made by the ribosomes within a cell. The enzymes that need to stay within the cell are made by the free ribosomes in the cytoplasm. The enzymes that are made to be exported to other cells are created by the ribosomes on the Rough E.R. The purpose of enzymes is to lower the activation energy (the energy that is put in at the beginning of the reaction of a reaction! therefore speeding the reaction rate up. "atalase is one of the enzymes found in the cells of the human body. #t is made up of five hundred amino acids in four polypeptide chains. #t is found in the perioxisome microbody in the cells of eu$aryotic organisms. The perioxisomes ma$e hydrogen peroxide (%&'& . #t is a byproduct of cellular respiration. This is very toxic to cells. That is why catalase is there. "atalase brea$s down the hydrogen peroxide before it damages the cell. The formula for the reaction of decomposition of hydrogen peroxide by catalase is: %&'& %&'& %&' ( '& %&' ( '&

)ubstrates are the substances that attach to the enzyme. They attach at the enzyme*s active site with induced fit. The combining of and enzyme and substrate re+uires ,T-. The reaction when an enzyme and a substrate .oin is reversible. The reaction can ma$e more products or it can ma$e more substrate.

Enzymes are affected by many things! such as salt concentration, p%, temperature, substrate concentration, product concentration, activators, and inhibitors. These things can cause the enzyme to denature (change shape . #f the enzyme denatures, then the substrate isn*t able to attach to the active site of the enzyme. The typical salt concentration for an enzyme is intermediate. This allows the substrate to attach to the enzyme correctly. #f the concentration is lowered the R groups of the enzyme attract to each other and pull the enzyme into a different shape (changing the active site . #f the concentration of salt is raised, then the active site of the enzyme is bloc$ed. Therefore the substrate can*t attach to the enzyme. Enzymes function best in an environment with a p% of seven. /hen the p% of the enzyme*s environment become more acidic (% ( ions increase the enzyme denatures. This causes the active site to change its shape and the substrate can*t attach. The same thing happens when the enzyme*s environment gains %( ions (becomes more basic . /hen heat is added to anything the molecules increase is movement. /ith an enzyme, when ten degrees "elsius is added the reaction rate is doubled. This will continue until optimum temperature is reached. Then the enzyme is destroyed. Enzymes denature at approximately forty0fifty degrees "elsius. Enzymes follow the 1aw of 2ass ,ction. This determines the rate and direction of an enzyme and substrate reaction. #f there is a high amount of substrate and a low amount of products the reaction will continue to ma$e more products. #f there is a low amount of substrate and a high amount of products, then the reaction will stop to get more substrate to continue the reaction. This is true except when products are immediately metabolized or exported from the cell. ,ctivators enable the substrate to fit the active site better. They increase the reaction. #nhibitors bloc$ the active site or cause the enzyme to denature. They slow down the reaction. #n this lab assaying is performed. ,n assay measures the amount of a substance that is left after a reaction. #n this case, hydrogen peroxide.

Hypothesis: The enzyme catalase will decompose %&'& (hydrogen peroxide best when its environment is ideal.

Materials: The materials used in the first part of exercise &, are 34m1 of 3.56 %&'&, one 54m1 bea$er, and 3m1 of fresh catalase. The materials used in the second part of exercise &, are one test tube, 5m1 of fresh catalase, a hot plate, 34m1 of 3.56 %&'&, and one 54m1 bea$er. The materials used in the third part of exercise &, are a potato, a $nife, a ruler, one 54m1 bea$er, and 34m1 of 3.56 %&'&. The materials used in exercise &7 are 34m1 of 3.56 %&'&, 3m1 of water, 34m1 of %&)'8 (3.4 2 , a stirring rod, two 54m1 bea$ers, 5m1 syringe, and 5m1 of &6 92n'8. The materials used in exercise &" are a 54m1 bea$er, 34035m1 of 3.56 %&'&, 34m1 syringe, and 5034m1 of &6 92n'8. The materials used in exercise &: are ;4m1 of 3.56 % &'&, ;4m1 of %&)'8 (3.42 , ;m1 of fresh catalase, six 54m1 bea$ers, three 34m1 syringes, six 34m1 bea$ers, and <4m1 of &6 92n'8.

Methods: Exercise &,: To observe the reaction of catalase and hydrogen peroxide, transfer 34m1 of 3.56 %&'& into a 54m1 bea$er and add 3m1 of fresh catalase. 'bserve the reaction and record results on Table 3. =ext, the effect of boiling catalase needs to be demonstrated. -lace 5m1 of fresh catalase into a test tube and boil it over a hot plate for 5 minutes. ,llow the boiled catalase to cool very well. Then place 34m1 of 3.56 % &'& into a 54m1 bea$er and add 3m1 of the cooled, boiled catalase. 'bserve the reaction and record results on Table 3. >inally, to see the presence of catalase in living tissue, cut 3 cm< of potato and macerate it. Then, place it in a 54m1 bea$er with 34m1 of 3.56 %&'&. 'bserve the reaction and record results on Table 3.

Exercise &7: This is the procedure for establishing a baseline. >irst, put 34m1 of 3.56 %&'& in a bea$er. ,dd 3m1 of water (instead of catalase . ,dd 34m1 of %&)'8 (3.42 . 2ix the solution well. Remove a 5m1 sample from the solution and place it into a 54m1 bea$er. ?sing the 5m1 syringe, add &6 92n'8 one drop at a time (swirling after each drop to the sample until a persistent pin$ or brown color is obtained. Record the results on Table &. Exercise &": This will determine how much %&'& will decompose spontaneously in an uncatalyzed reaction. -lace 34035m1 of % &'& in a 54m1 bea$er and store it uncovered for approximately &8 hours :etermine the amount of %&'& remaining after &8 hours by running a baseline test. ?se a 34m1 syringe to get 34m1 of &6 92n'8. ,dd the 92n'8 to the solution one drop at a time (swirling after each drop until a pin$ or brown color is permanently obtained. Record the results in Table <.

Exercise &:: #f a day or more has passed since exercise &7 has been performed, reestablish the baseline by performing exercise &7 again. Record the results in Table 8. This exercise will determine the amount of %&'& is disappearing over 34, <4, ;4, 3&4, 3@4, and <;4 seconds. >irst, place 34m1 of 3.56 %&'& in six 54m1 bea$ers. 1abel the bea$ers 34, <4, ;4, 3&4, 3@4, and <;4 seconds. 'btain ;m1 of fresh catalase and ;4m1 of % &)'8 (3.42 . ,dd 3m1 of catalase to the 34 second bea$er with one syringe. ,t 34 seconds, add 34m1 of %&)'8 (3.42 with another syringe. >or each of the times, repeat the adding of the catalase and % &)'8. ,llow the reactions to proceed for <4, ;4, 3&4, 3@4, and <;4 seconds respectively. ,fter the times have passed remove a 5m1 sample from each of the six bea$ers and place each of the samples into a separate 34m1 bea$er. ?se 5ml of &6 92n' 8 for each of the six bea$ers to determine the amount of %&'& that is left after the reaction. ,dd 92n'8 to the samples one drop at a time (swirling after each drop until a pin$ or brown color is permanently obtained. Record results in Table 5. Araph the results.

Results:

Table 3 Enzy e Acti!ity

Acti!ity Enzyme ,ctivity Effect of Extreme Temperature -resence of "atalase

"#ser!ations 7ubbles =othing 7ubbles

Ta#le $ Esta#lishin% a &aseline

#nitial Reading

'olu e 5m1

>inal Reading

3.;m1

7aseline (final volume B initial volume

<.8m1

Ta#le ( Rate o) Hydro%en *ero+ide Spontaneous ,eco position

#nitial 92n'8 >inal 92n'8

'olu e ;m1 3m1

,mount of 92n'8 used after &8 hours ,mount of %&'& spontaneously decomposed (ml baseline B ml after &8 hours -ercent of %&'& spontaneously decomposed (ml baseline B ml after &8 hoursC baseline

5m1 3.;m1 5<6

Ta#le Esta#lishin% a Second &aseline

#nitial Reading

'olu e 5m1

>inal Reading

3m1

7aseline (final volume B initial volume

8m1

Ta#le . Rate o) Hydro%en *ero+ide ,eco position #y Catalase

&aseline 5Mn"-

12 8m1

(2 8m1

Ti e / Seconds0 32 1$2 8m1 8m1

142 8m1

(32 8m1

Initial !olu e 5Mn"6inal !olu e 5Mn"A ount 5Mn"- used /#aseline 7 )inal0 A ount H&"& used /5Mn"- 7 initial0

5m1 &m1 <m1 3m1

5m1 <m1 &m1 &m1

5m1 8m1 3m1 <m1

5m1 8.&m1 .@m1 <.&m1

5m1 8m1 3m1 <m1

5m1 8.&m1 .@m1 <.&m1

18

,eter ine the initial rate o) the reaction and the rates #et9een each o) the ti e points8

4034 0 .3 340<4 0 .45 <40;4 0 .4< ;403&4 0 .44< 3&403@4 0 .44< 3@40<;4 0 .443

$8 :hen is the rate the hi%hest; E+plain8

They are the highest at the beginning of the reaction because! less hydrogen peroxide had been spontaneously decomposed. Therefore, there was more to be bro$en down.

(8

:hen is the rate the lo9est; 6or 9hat reasons;

They are the lowest at the end of the reaction because more hydrogen peroxide has been decomposed.

-8

E+plain the inhi#itin% e))ect o) sul)uric acid on the )unction o) catalase8 Relate this to enzy e structure and che istry8

The acid lowered the p% and the change made catalase gain % ( ions which caused the active site to change shape.

.8

*redict the e))ect lo9erin% the te perature 9ould ha!e on the rate o) enzy e acti!ity8 E+plain8

#t would decrease the reaction because changing the temperature causes the enzyme to denature. This causes the active site to change place, which slows down the reaction.
38 ,esi%n a control e+peri ent to test the e))ect o) !aryin% pH< te perature< or enzy e concentration8

1abel < test tubes 4o", rt, and 344o". -lace an e+ual amount of enzyme in each tube. -ut the 4o" on ice, leave the rt out and boil the 344o" tube. 'bserve the changes in all < tubes.

Error Analysis: )ome possible errors that could have occurred in this lab include ta$ing inaccurate measurements of the substances used, ma$ing wrong calculations, and the times of testing the experiments could have been delayed (ex: instead of ;4 seconds it too$ place at ;5 seconds .

Conclusion: The enzyme catalase is created to brea$ down %&'&. #f the environment of catalase is changed in any way! such as p% and temperature! then it can not do its .ob of brea$ing down %&'&. This is proven when catalase bro$e down %&'& in &, and produced water and oxygen when it wasn*t boiled. /hen the catalase was boiled, it had been denatured! therefore it wouldn*t brea$ the %&'& down. #n &: the sulfuric acid $ept catalase from brea$ing down %&'& also. #t lowered the p% of the solution so the catalase denatured. Dou can tell by loo$ing at Table 5. The lower times have less %&'& used than the higher times. The higher times had more time with out the %&)'8, so the catalase was able to brea$ down more % &'&. The rates of the reaction in this lab were very high at the beginning. Then, they started to slow down. This is because at the beginning of the reaction there is more %&'& to decompose. Towards the end of the reaction there is less to decompose, so the rate slows down +uite suddenly.