Catalytic Activity of Enzymes

Introduction

Enzyles are biological molecules that catalyze (speed up) chemrcal reactions. You could call enz;anes the “Builders and Do-ers” m the celi: without them, iife could not occur. Every ceI1 makes hundreds of different enzyxes to carry out the reactions necessary for 1ife. Fortunately for the ce1l, enzlmes are not used up when they calaltlze a reaction. but can be used over and over.

The DNA in each ce11 encodes all the information needed to make its many different enz)rmes. Enzlnnes are relatively large molecules of protein. They are produced u,henever tire cel1″senses” a need for that parlicular enzyne; that is, whenever a job needs to be done in the ce ll which only that enzyne can do it.

The molecule (or molecules) on u,hich an enzyne acts is called its substrate. Enzla:nes are said to be very “specific,” meaning that they recognize only one substrate (or a fei,r, closeiy related substrates) and convert it into a specific product. You could say that each enzl.me can do only one tlpe ofjob. Each en4,rne is specrfic because it is folded urto a particular three-dimensional shape. Wrtirur the folds of each enzyle is the active site, the place u,here the substrate fits and where the chemical reaction takes place.

Enzynes work very quickly, often catalyzing thousands of reactions per minute. The rate at which an ervrme works is influenced by many factors including temperature and pH. Enzl.mes have a temperature and pH at which they work best, and if an enzyne is exposed to extremes of heat or pH it won’t rvork at a1ll The interactions that hold the protein in its particular shape become disrupted under these extreme conditions, and the 3-dirnensional structure unfblds. In this case, the enzyxe is said to be denatured. Other important factors that influence enz),rne activity are the concentration of substrate and the concentration of enzyle. Up to a point, the more substrate that is present, the fuster the reaction. Hou,ever, u,hen the substrate concentration is so high that an enzyne is workilg as fast as it can, fui1her increases of substrate concentration will have no effect on the rate of product formation.

Background

The enzyne that you will study in this experiment is called “catalase.” Its job is to break down its substrate hydrogen peroxide (HZOZ,), wirich is a naturaily occuning poison. Vy’ithout catalase, HZOZ

could krll the cell. The reaction calalyzed by catalase is:

2HyO2+2H2O+Oy

The products remaining alter catalase does its job are oxygen gas and water; two very non-poisonous molecules.

CORNELL”. HHMI

wru A,M

O2OO8 CIBT A Study of Catalase – Teacher Section Page 1

 

 

ln the home and hospita| h,vdrogen peroxide is used as an antiseptic to ciean out wounds. Have you et,er noticed that when hydrogen peroxide is su,abbed on a cut it bubbles? This is because enz),rnes ln the cut fiom your body and from infecting bacteria catallze the rapid degradation of h,vdrogen peroxide into water and oxygen. The bubbles are oxygen.

Catalases are very corrrroll. They are found in almost all celis that grorv in oxygeu, inciuding potato tubers. In this experiment, a blender is used to grilrd up a potato in water to release the catalase from the potato cells. The ground-up potato is filtered through cheesecloth to separate potato skin and celI debris from the liquid which contains n’lost of the cell’s enz),rnes, including catalase. To actualiy measure the catalase activify, small disks are dipped rnto the potato cell extract” \&en this enzyrne-contaimng disk is placed in a solution of hydrogen peroxide, the enzyme begins to work. As rhe catalysis occurs, oxygen is produced, and bubbles of the gas become trapped in the fibers of the disk. When there are enough O2

bubbles. they 1ift the fi1ter to the surface. The speed with which the 02 is produced depends both upon hou,’much enz)4xe is present and on the concentration of the irydrogen peroxide. The more enzyme, the fuster the product (O2) is made. SLmilarly, the higher the concentration of the substrate, hydrogen

peroxide, tire faster the product is made. You can see u,hat happens u,hen you vary either the concentration of enzlnne or the concentration of the hydrogen peroxide.

To do this experiment, five of the teams of students will carry out one version of the experiment using low, medium, and high enz)rne concentration and a constant 1evel of substtate, 1.0%H2a2. Then, the other six teams will do another version of the experiment using low, medium, and high H2O2 concentration u,ith

cell extract diluted to 600/o of its original concentration. At the end of the 1ab, experimental results will be pooled and the class as a u,hole will generate data showing the relationship befiveen catalase activity and both enzl,rne and substrate concentrations.

A third experiment dealing with the effect of pH is provided. This porlion could be performed easily by one or nvo teams of students. It aiso lends itsclf nicely to a teacher demonstration for the entire class.

For an experiment to be meaningful, there must be controls. Three controls imporlant to this lab rviil be demonstrated by your teacirer:

Control #1: A paper disc that has not had potato extract added to it is dipped in H2O2.

Control #2: A paper disc that has been dipped in potato extract is placed in a beaker of water.

Control #3: A paper disc that has first been dipped in boiled potato extract and then placed into a beaker of H2O2.

Observe and record on page 8 what occurs as each control experiment is dernonstrated.

Why is each control important?

What does each control experiment show you?

O2OO8 CIBT Catalytic Activity of Enzymes * Student Section Page2

 

 

N{aterials

Your tearn u,ill need the following suppiies:

. potaio extract (prepared by your teacher)

. 1000 mi flask rvitir distilled/deionized/dechlorinated water o I – 250 ml beaker for potato extract . 200 ml o/o HZOZ solution for the first parl of the experiment

. 3okT12A2 solution to dilute for second part of the experimerrt

. 100 ml graduated cyiinder r g – 100 m1 beakers

. forceps . 40 filter paper disks

. paper towels . stopwatch, if available

. 6A0 catalase . calculator

Experimental Procedure for Teams Working $,ith Various Catalase Concentrations

1. Making the potato extract:

. Watch vour teacher prepare the potato extract as {bllows:

. Cut clean potatoes into chunks (allow one potato per team of students)

. Place the potato chunks in tire blender and add 200 ml of buffer per potato.

. Puree in the blender.

‘ Pour potato puree through four layers of cheesecloth placed in funnel. Collect as much fluid as possible. This fluid contains the enzl.Tne catalase. among many other things that were stored inside the cells of the potato.

‘ Add enough distilled water to bring the final volume to 200 n.rl per potato. Swirl the flask to mix the solution. This willbe arbitrarily designated as “100%” catalase extract. (Each team should have a 2-50 mi beaker containing 200 m1 of 100% catalase.)

2. Together wrth your pafiners, prepare your enz),rne concentrations in the beakers.

. Labei the beakers with tape and pen: 20oh, 40oA, 600 , 80o , and 100%.

‘ Make the appropriate dilutions. For example, if you are doing test #1(20%), measure 8.0 mi of the potato extract using the graduated cylinder and pour into the beaker.

. Rinse the graduated cylinder, then add 32.0 m1 of distilled H2O and stir well with the stirring rod.

O2OO8 CIBT Catalytic Activity of Enzymes – Student Section Page 3

 

 

. Make the rest of the enzyme solutions using the chart beiow as a guide.

Test Extract Concentration Volume of En4mre Volume of Water

#1 20% 8ml 32ml

11^l 40% 16 ml 24 nl

ItJ 60% 24 ml 16 ml

!n t++ 8A% 32ml 8ml

ra0% 40 ml 0ml

. Obtain the flask of 1o/o h,vdrogen peroxide if it is not at your tabie. This is tire substrate for this part of the lab.

3, Now you are ready to begin measurilg the effects of enzyrne concentration on enzyme activity.

. Pour 30 ml of the 1% HZOZ solution into a clean beaker, iabeled “reaction beaker.”

. Pick up a paper disk with a clean forceps. Using the forceps, dunk the disk in your en44ne extract for 5 seconds, until the disk is uniformly moistened but not beaded with shiny drops of liquid”

. Drain it on a piece of paper towei for 5 seconds to remove oxcess enz).rne frorn the disk.

4. The reaction is now ready lo be starled and timed.

. Using forceps. place the filter disk (containing the enzynes) onto the bottom of the “reaction beaker” contauing 1% hydrogen peroxide.

. One person should watch the cloclc/stopwatch, another watch the rising disk. Stop timing as soon as disks have completely ‘lifted off the bottom of the reaction beaker.

. Watch the filter disk. You should see tinybubbles of oxlgen being released as the hydrogen peroxide is broken into water and oxygen by the catalase.

. Record the time in seconds for each trial on the char1. Be precise in your timing and recording.

. Remove the disk and discard rt.

5. Obtain another disk and repeat steps 3-4 exactly as done above.

6. Repeat the experiment a thil’d tilne exactly as above: now you have “triplicate” measurements of the rate of oxygen productio n in lok HZOZ at each particular enzyxe concentration. Average these 3

values and record in the charl below. Repeat this procedure for all the concentrations of enzyne.

O2OO8 CIBT Catalytic Activity of Enzymes – Student Section Page 4

 

 

Data Table / Enzyme Concentration

Test Number

Triall Trial2 Trial 3 Team Average

Class Average

1

20% 7 ,39/’ -?jh /’ ,””,.,,,… /a^n/>’ cn1-r/L ) 2

40% J/tL t L,//y, -)

(L- L./,/

4< /qF

L. i|b I 1 J

60% .7 s/bb , .”$L/qs ,.u? 1) > 1

80% t , (/za I’i-Qt,,/:- I . t–7 *?1 5

100% .0q I /1 | . , r”/, i,2 77)

1. Clean up all materials!

Experimental Procedure for Teams Working with Various Hydrogen Peroxide Concentrations

I . Label the clean beakers with the percent h,vdrogen peroxide that will be used in this part of the experiment: 20 , 1.5o/o,1.Ao ,0.80 ,0.60 and 0.30h. Together with i,our partner, prepare your particular substrate concentrations il each of tire labeled beakers. For example, for test #1, measure 20.0 m1 of the concentrated HZaZ using the graduated c,vhnder and pour urto the beaker. Add 10 ml

of distilied u,ater to the -sraduated

cyiinder and pour into the hydrogen peroxide. Stir well with the strn-urg rod. Rinse the graduated cyhnder. See chart for proportions of hydrogen peroxide and water to mix for each dilution. Place all of the beakers on the table in front of you in order fiom lowest to highest concentration of hydrogen peroxide.

Test # Substrate Concentration Volume ofL120y Volume of Water

1 2.4% 20 ml 10 ml

2 15% 15 ml 15 ml

aJ 1.0% 10 ml 20 ml

4 0.8% 8ml 22ml

5 0.6 % 6ml 24 ml

6 0.3 % 3ml 27 ml

Next, obtain your 60% catalase solution.

Using forceps, dip a disk in the 60% potato extract for 5 seconds, 1et it drain on a paper towel for 5 seconds. Then, using forceps, place the filter (contarning enzynes) on the bottom of the “reaction” beaker Q.A%HZO).

Time how long it takes the disk to rise from the bottom of the beaker to the top of the liquid. Be sure

2.

3a.

b.

CI2008 CIBT Catalytic Activitl’ of Enzymes – Student Section Page 5

 

 

that the disk is placed at the bottom of the irydrogen peroxide before you start to time the experiment.

c. Record the time in seconds in the appropriate space on the chart that fo11ows.

4. Obtain another disc and repeat steps 3a – 3c exactly as before,

5. Repeat the experiment a third time. Now you have triplicate measurements of the rate of oxyger-r production. Average these three values and record on the chart.

6. Repeat this procedure for all of the concentrations ofH2O7.

Data Table / Substrate Concentration

Test Number

Triall Trial2 Triai 3 Team

Average Class

Average

I ggaVo 2 .a’l ,

‘i “/,t, -‘l mnt !L t. I i /qz – 5>fns /*r4 r z 4A% 3,9″,’ 7,’tt /ii, 4,atl?q,I -l Z J , 3

69t7, ” ‘a ‘7

( rr’.’ 1,teft=; ( 7, i^tat) tL i lL t ^r? qs ),/ t 4 80% b,t 7 b , ,:o7 $,1o ly7 * 5,7o! ”, ya Itl s 5

IOO%.S.5 ‘/ 1,zc I d,?$f :ts $,<t%f tuq ]11 ) ,?/. $ , ‘i.*l’*.ae 4 no /(ilairiv ^ ,l I J|u t1. Ciean up all materials! I

O2OO8 CIBT Catalytic Activity of Enzymes – Student Section Page 6

 

 

After the Experiment

Controls

A. &hat is the function of a control?

For control #1, a fiter paper saturated with water rather tiran potato extract was placed tn a beaker of I%H7O2. Horv long does it take for the filter to lift off.r Explairi the significance of the result:

For control #2, a piece of filter paper was saturated with potato extract and then placed in distilied

water. How longdid it take for the filter to lift off.r Explain the significance of the results:

For control #3, 100% catalase was boiled. A filter paper was then saturated with this extract. The disk containhg the extract rvas then piaced in a beaker of 1o/oHZOZ. How long did jt take for the

filter to lifl off? Explain significance of the resuits:

B I . pool your results with those of the rest of the class, record belorv and fi1l in the class average portion of the table for your experiment and also for the other experiment.

O2OO8 CIBT Catalytic Activity of Enzymes – Student Section PageT

 

 

test team 1 teamZ team 3 team 4 team 5 team 6 team 7 team 8

1:20oh >L+ 5 L-)

2:400/o Llt t r (o/1 -LJ (

3:60% Tqt aq 4:80oh

O4/i C

A< (

5:100% ?a 1 7Zt 4/- (-

Data Table for Enzyme Concentration (mean for each test)

2.

Data Table for Substrate Coneentration (mean for each test)

test team I team2 team 3 leam 4 team 5 teasa'{ t?j.fn I ftffi 1:2.0% )(lclt-f ,TF

2: 1.50k 6r -) -n/ () c I Q0, 3: l.0o/o ‘bt

*L'{ . ( tn

4:0.8o/o r{* ?”4*i l?>

5: 0.60/o z{1)) 4t s 7t6 6: 0.3o/o

l-) lL- 9 3zs Lfzs

Plot both the team and the class averages on graph paper. The frst graphs should be “concentration of enz),rne vs. time of reaction.” The x-axis is designated concentration, starting at point 0. The y- axis is 1/t, so you have to do the math on this before you graph your data. The second graph should

be “concentration of substrate vs. time of reaction.” The x-axis is designated concenkation, starting at point 0. The y-axis is again 1/t, so do the math first.

O2OO8 CIBT Catalytic Activity of Enzymes – Student Section Page 8

 

 

-?. Discuss three factors that influence the raie of enzFne action:

4. \4ry did you do the experiment in tripiicate?

02008 CIBT catalytic Activity of Enrymes – student section Page 9

Pabio/Meded 536 Protein BLAST and Motifs – Homework Purpose: Become familiar with Protein BLAST queries of the NCBI non-redundant databases, using the human acyl-CoA binding protein as a query. To examine the range of BLAST hits, we will restrict the output to C. elegans sequences. We will examine the BLAST alignment outputs for a number of the top BLAST hits and relate the alignments to the amino acid conservation that is obvious from multiple alignment of the human, bovine, duck and yeast ACBP family members (see alignment link in question 7). Examine this alignment and identify recognizable conserved motifs which you will use as the basis for determining homology of the C.elegans sequences with the ACBP protein family. Initially you will evaluate the C.elegans hits by eye, looking for conservation of your chosen motifs. Provide your assessment and indicate your reasons. Then you will take each C.elegans sequence you believe has homology to the ACBP family and do a new protein BLAST search against the Conserved Domain database (you can do this analysis as a protein BLAST search against the non-redundant database, since your protein will be automatically searched against the Conserved Domain database – or you can link to the Conserved Domain database and do the search directly from the links at this site.) You will do two different analyses using the information from the Conserved Domain search. First, reevaluate your assessment of the homology of the C. elegans sequences to the ACBP family. Second, determine whether these C.elegans sequences have any other motif recognized by the conserved domain search. You will then use the multiple repetition PSI-BLAST search, which computes an ACBP-specific scoring matrix based on the aligned ACBP sequences, in an attempt to identify additional C.elegans sequences that are distantly related to the ACBP family. Using all this information you will determine whether C.elegans has an ortholog of human ACBP, providing your criteria. In addition, you will determine how many of the C.elegans sequences contain a recognizable ACBP domain. From this make a guess as to the evolution of the C.elegans ACBP family. Finally, you will use PHI-BLAST, to combine a pattern search (A pattern you have determined from the alignment linked in question 7) with BLAST to again search the non-redundant protein database (limited to C.elegans sequences) to evaluate how PHI-BLAST can be used to detect distant similarities. The first extra credit question will test your ability to compare BLAST results for ACBP family members in humans and drosophilia to determine the evolutionary history of ACBP in these organisms and whether they differ from the C.elegans ACBP family members. The second extra credit question will introduce you to phylogenetic analysis.

1. Retrieve the Acyl-CoA Binding protein (human) sequence (87 amino acids) from the class sequence link or this link and do a standard Protein BLAST search against the NCBI Non-Redundant protein database. IMPORTANT- limited for only the Caenorhabiditis elegans sequences. (In the organism box, start typing “caen” and then choose Caenorhabiditis elegans from the list. – your analysis should only find a restricted number of C. elegans hits). Prepare a table giving the accession numbers, number of amino acids in the protein and Evalues for the

 

 

top 10 BLAST hits. How many ACBP family members are detected? What was your criteria? (Evalue score? Sequence title? (10 pts)

2. Examine the alignments of the top 10 C.elegans hits resulting from your search above. Using your knowledge of the conservation of motifs in the ACBP gene family (see ACBP conservation alignment) determine whether any of the alignments reveal homology to the conserved ACBP motifs (Two motifs YxxYKQA and KWxAW are provided in the ACBP alignment) ? In your table in question 1, add a column for each motif and indicate whether either either motif is found in these 10 C.elegans sequences (Y/N/?). How many ACBP-related sequences do you think are in C.elegans? (10 pts)

3. Take each of the C. elegans in question 2 that you have determined to be related to ACBP (those labeled with a Yes in your table) and determine whether they have a ACBP motif that is detected by the Conserved Domain Search (use each sequence in a BLAST search against the Conserved Domain database). Add a column in your table above (BLAST-CD) and indicate whether there is a conserved ACBP domain detected by BLAST (Y/N). How does this compare to your analysis of the sequence alignments? (10 pts) Do any of these proteins have more than one recognizable functional domain (The BLAST search against the CD database will show you this)? Add a column to your table (Other domains) and indicate the domain name (10 pts). Do the same analysis for the remaining BLAST hits of the top 10 that you determined in question 1 and 2 to not be related to ACBP. Indicate the domains detected by BLAST for these sequences and indicate these results in your table (ACBP? Other?). (10 pts). How many ACBP-related sequences do you now think are in C.elegans?

4. Use the ACBP human protein sequence to do a PSI BLAST search of the non- redundant protein database, limiting to C.elegans.. The PSI BLAST results will select all protein matches above a selected E-value to be used in determining a new ACBP-specific scoring matrix. Use the default setting and perform the second iteration of the PSI-BLAST search. Add a new column to your table (PSI- E) and indicate the new E values determined for your top 10 C.elegans hits in your table. Is there a new C.elegans sequence that has a significant E-value that was not readily detected in the original protein BLAST search? From this analysis, how many C.elegans sequences do you think are related to human ACBP? (10 pts)

5. Does C. elegans have an ortholog of ACBP? State your reasons and provide your criteria and ID. An Evalue alone is not sufficient. (10 pts).

6. Provide a short explanation regarding the evolutionary history of the ACBP homologs in C. elegans, discussing gene duplication, conservation of structure and function, and structural similarities between homologs –only based on the data from your analysis (20 pts).

7. Determine an amino acid pattern that is conserved in the N-terminal domain of the ACBP orthologs in the linked alignment which is also found in your “top” choice of the ACBP ortholog in c.elegans (CLICK HERE for alignment). What is your pattern? Do a PHI-BLAST search using the human ACBP as the probe, and insert your pattern to be searched. Limit the search to C. elegans sequences. How many of the C.elegans ACBP-like sequences contain your pattern? Would you

 

 

consider this pattern specific to ACBP orthologs? Do a PHI-BLAST search with this pattern using the C.elegans ACBP “ortholog” as a probe, limiting your search to human sequences. How many “different” (ie coming from different genes/chromosomes) human sequences match this pattern? Discuss, especially in relationship to orthology between the human and c.elegans sequences. (10 pts).

8. Extra credit – can you determine a pattern that is found in more than 4 of the C.elegans ACBP-like sequences? What is it? Do a PHI-BLAST search with the pattern and one of these four sequences as query, limiting your search to C.elegans. How many C.elegans ACBP-like sequences is it found in (10 pts). Do the same search but limit to human sequences. Are there any human ACBP-like sequences that contain this pattern. Provide one example.(10 pts)?