Lab Topic 2

Enzynes

Laboratory ObjectivesAfter completing this lab topic, you should be able to:1. Define enzyme and describe the activity of enzymes in cells.

Z. Differentiate competitive and noncompetitive inhibition.3, Discuss the elfects of varying environmenral conditions such as pH and

temperature on the rate of einzwe actlvlt)¿4. Discuss the effects of varying enzyme and substrate concentrations on

the rate of enzyme activity.5. Discuss the scientific process, propose hypotheses, and make predic-

tions based on hypotheses.6. Practice scientific persuasion and communication by constructing and

interpreting graphs of enzpe activity

IntroductionLiving cells perform a multitude of chemical reactions very rapidly becauseof the participation of enzymes. Enzymes are biological catalysts, com-pounds that speed up a chemical reaction without being used up or alteredin the reaction. The material with which the catalyst reacts, called thesubstrate, is modified during the reaction to form a new product (seeFigure 2.1). But because [he enzyme itself emerges from the reactionunchanged and ready to bind with another substrate molecule, a smallamount of en4rme can alter a relatively enormous amount of substrate.

The active site of an enzyme will bind with the substrate, forming theenzyme-substrate complex. It is here that catalysis takes place, and whenit rs complete, the complex dissociates into enz)¡me and product or products.Enzymes are, in part or in whole, proteins and are highly specific in func-tion. Because enzyrnes lower the energy of activation needed for reactionsto take place, they accelerate the rate of reactions. They do not, however, deter-mine the direction in which a reaction will go or its final equilibrium.E ntal con-o 1ilå.:iä:iff"dshapeo chemicals may also bindto an enzyme and modify its shape. Chemicals that must bind for the en4.ryneto be active are called aótivatorÂ. cofactors are nonprotein subs.tances thatusually bind to the active site on the en4rme and are essential for the errz.rnre

33

Please read through this lab exercise. Where responses are required, please use the separate handout I gave you in class.

IB Biology C

34 Lab Topic 2:Enzymes

Figure 2.1.EnzFe activity. A substrate orsubstrates bind to the active site ofthe en4¡me, forming the en4¡me-substrate complex, which thendissociates into enzyme and product(s).The enz)¡me may catalyze the additionor removal of a molecule or a porlionof a molecule from the substrate toproduce the product (a), or theenzqemay catalyze the splittingof a substrate into its componentsubunits (b).

Enzyme-substratecomplex

Enzyme-substratecomplex

Substratesplits

/

Products

/j )

Productsir)

-Enzyme is ready tocatalyze another reaction

-

-

-

a.

b.

to work. Organic cofactors are called coenzymes, but other cofactors maysimply be metal ions. Chemicals that shut off enzqe activity are calledinhibitors, and their action can be classified as competitive or noncompetitiveinhibition.Review Figure 2.I, illustrating enzyme activit)¿ There are two \Mays to mea-sure enzyrne activity: (1) Determine the rate of disappearance of the substrate,and (2) determine the rate of appearance of the product.

In this laboratory, you will use both methods to investigate the activity oftwo enzymes, catechol oxidase and amylase. You will use an inhibitor toinfluence the activity of catechol oxidase and determine if it is a competi-tive or noncompetitive inhibitor. Additionally, you will investigate the effectof changing environmental conditions on the rate of amylase activiq¿

EXERCISE 2.IExperimental Method and theAction of Catechol Oxidase

Materialstest-tube rack3 small test tubessmall ParafilmrM squarescalibrated 5-mL pipette3 calibrated l-mL pipettesdisposable pasteur pipettes

pipette fillerpipette bulbdistilled or deionized (DI) waterpotato extractcatecholdisposable gloves (optional)

You will not actually carry out this lab. This is a dry-lab, meaning you will get the data you might have obtained by following the procedure, and then you will analyze those data to learn about the concepts being studied.

Lab Topic 2: Enzymes 35

IntroductionThis exercise wrll investigate the result of catechol oxidase activit)¿ in the pres-

alyzes the removal of electrons andmpound found in plant cells. Catecholent product. The hydrogens combine

with oxygen, forming water (Figure2.2). The pigment products are respon-sible foiThe darkening of fruits and vegetables, such as apples and pota-toes, after exposure to air.

In this exercise you will use an extract of potato tuber to test for the pres-ence of catechoi oxidase and to establish the appearance of the productswhen the reaction takes Place'

oOH Catechol

oxidaseU

+ 1lz O" + Hro

Catechol Benzoquinone

Figure 2.2.The oxidation of catechol. In thepresence of calechol oxidase, catecholis converted to benzoquinone.Hydrogens removed from catecholcomblne with oxygen to form \Mater.

QuestionRemember that every experiment begins with a question, Review the infor-mation given above about the activrty of catechol oxidase. You will be per-forming an experiment using potato extrac[.Formulate a question about catechol oxidase and potato extlact. The ques-tion may be broad, but it must pïopose an idea that has measurable andcontrollable elements.

HypothesisConstruct n or absence of catechol oxidase inpotato extra must be testabie. lt is possible foryou to prop but all must be testable'

PredictionPredict the result of the experiment based on youl hypothesis. Tô test forthe presence or absence oftatechol oxidase in potato extract, your predic-tion would be what you expect to observe as the result of this experiment(iflthen).

Formulating a research question is a crucial step in an investigation. Write the appropriate question on the separate answer sheet

Write your hypothesis on the answer sheet

Write your prediction on the answer sheet (make it an if-then statement)

36 Lab Topic 2: Enzymes

Catechol is a poison! Avoid contact with all solutions. Do notpipette any solutions by mouth. Wash hands thoroughly aftereach experiment. lf a spill occurs, notify the instructor. lf theinstructor is unavailable, wear disposable gloves and use drypaper towels to wipe up the spill. Follow dry towels with tow-els soaked in soap and water. Dispose of all towels in the trash.

Procedure

1. Using Table2,1, prepare the three experimental tubes. Note that alltubes should contain the same total amount of solution. Do not cross-contaminate pipettes! After each tube is prepared, use your finger tohold a ParafilmrM square securely over the tube mouth and then rotatethe tube to mix the contents thoroughly Use a fresh square for each tube.

Table 2.1Contents of the Three Experimental Tubes

2. Explain the experimental design: What is the purpose of each ofthe three test tubes? Which is the control tube? Is more than one controltube necessary? Explain. Which is the experimental tube? Why is an addi-tional 0.5 ml of distilled water added to tubes I and 3, but not tube 2?

3. Observe the reactions in the tubes, and record your observations inthe Results section below Explain your conclusions in the Discussionsection.

ResultsDesign a simple table to record results (Table 2.2)

Table2.2Results of Catechol Oxidation Experiment

Tube Distilled Water Catechol Distilled Water Potato Extract

I 5ml 0.5 ml(I0 drops)

0.5 ml(10 drops)

2 5ml 0.5 ml(10 drops)

0.5 ml(10 drops)

3 5ml 0.5 ml(10 drops)

0.5 ml(10 drops)

Tube 1. (Write on answer sheet)Tube 2. Tube 3.

(I designed it for you)

(Write on answer sheet)

(Note: I will give you these data in class)

DiscussionExplain your results in terms of your hypothesis.

EXERCISE 2.2Inhibiting the Action of Catechol Oxidase

IntroductionThis exercise will investigate the inhibition of en4tme activity by specificchemicals called inhibitors. The specific inhibitor used will be phenyl-thiourea (PTU). To be active, catechol oxidase requires copper as a cofac-tor. PTU is known to combine with the copper in catechol oxidase andinhibit its en4¡matic activityAn inhibitor molecule affects aî enzqe in one of two ways. Competitiveinhibition takes place when a molecule that is structurally similar to thesubstrate for a particular reaction competes for a position at the active siteon the enzyme. This ties up the errzqe so that it is not available to thesubstrate. Competitive inhibition can be reversed if the concentration of sub-strate is raised to sufficiently high levels while the concentration of theinhibitor is held constant (Figure 2.3).

In noncompetitive inhibition, the inhibitor binds to a paft of the enz)tmethat is notthe active site. ln so doing, it changes the nature of the en4tmeso that its catalytic propenies are lost. This can happen in two ways. Eitherthe noncompetitive inhibitor itself physically blocks the access to the activesite, or it causes a conformational change in the protein, thus inactivatingthe active site. ln noncompetitive inhibition the inhibitor can become

Materialstest-tube rack3 small test tubessmall ParafilmrM squarescalibrated 5-mL pipette4 calibrated 1-mL pipettesdisposable pasteur pipettes

d.

pipette bulbdistilled waterpotato extractcatecholphenylthiourea (PTU)disposable gloves (optional)

I Substrate

Active siteI Figure 2.3.

Action of a competitive inhibitor.(a) Substrate normally can bind tothe ac[ive site of an enz¿rrre,(b) A competitive inhibitor mimicsthe substrate and competes for theposition a[ the active site on the erøyme.

Y

Competitiveinhibitor ,_-t

b.

Lab Topic 2: Enz\¡mes 37

(Write on answer sheet. Sentence 1: Reflect on your hypothesis to write a conclusion. Sentence 2: Cite your data to support the conclusion.)

38 Lab Topic 2: Enzymes

Substrateunbound, reversing the inhibition. However, unlike Çompetitive inhibition,adding additional substrate will not reverse the inhibition. (Figure 2.4.)In the following experiment, you will determine if PTU is a competitive ornoncompetitive inhibitor.

I

Enzyme QuestionPose a question about the activity of PTUNoncompetitive

inhibitor

Figure 2.4.Action of a noncompetitiveinhibitor. The noncompet"itiveinhibitor binds to the enz)'rne ata location away from the active site,either blocking access to [he activesite or changing the conformation ofthe enzyme, rendering it inacl"ive.

HypothesisHypothesize about the nature of inhibition by PTU

PredictionPredict the results ol the experiment based on your hypothesis (iflthen)

Procedure

PTU and catechol are poisonsl Avoid contact with solutions.Do not pipette any solutions by mouth. Wash hands thor-oughly after the experiment. lf a spill occurs, notify theinstructor. lf the instructor is unavailable, wear disposablegloves and use dry paper towels to wipe up the spill. Followdry towels with towels soaked in soap and water. Dispose ofall towels in the trash.

1. Using Table 2.3,prepare three experimental tubes. Be sure to add solu-tions in the sequence given in the table (water [iTSt, potato extract next,PTU next, etc.). Cover each tube with a fresh ParafilmrM square and mrx.

Table2.3Contents of the Three Experimental Tubes

TubeDistilledWater

PotatoExtract PTU

Distilled.Water Catechol

1 5mL 0.5 mL 0.5 mL 0.5 mL 0.5 mL2 5mL 0.5 mL 0.5 mL lmL3 5mL 0.5 mL lmL 0.5 mL

Write your answer on the answer sheet

Write your answer on the answer sheet

Write your answer on the answer sheet

Lab Topic 2: Enzymes 39

2. Which test tube is the control?

3. Why was [he concentration of catechol increased in test tube 2?

4. Why should the catechol be added to the test tubes last?

5. Record your observations in the Results section, and explain your resultsin the Discussion section.

ResultsDesign a table to record your results (Table 2.4)

Table2.4Results of Inhibition Experiment

Discussion1. Explain your results in terms of your hypothesis

2. One member of your team is not convinced that you have adequatelytested your hypothesis. How could you expand this experiment to pro-vide additional evidence to strengthen your conclusion?

Write your answer on the answer sheet

Write your answer on the answer sheet

Write your answer on the answer sheet

Write your table on the answer sheet

Write your answer on the answer sheet

(Write on answer sheet. Sentence 1: Reflect on your hypothesis to write a conclusion. Sentence 2: Cite your data to support the conclusion.)

(Note: I will give you these data in class)

IB Biology C / Sanderson Your Name__________________________

Lab Topic 2: Enzymes - Answer Sheet

Exercise 2.1Research Question:

Hypothesis:

Prediction:

ProcedureProcedure explanation:

Results Title:

Discussion:

Tube 1.Tube 2. Tube 3.

Exercise 2.2Research Question:

Hypothesis: PTU is a: ______________________

Prediction: If PTU is a ____________ inhibitor then:

Procedure2. Which test tube is the control?

3. Why was the concentration of catechol increased in tube 2?

4. Why should catechol be added to the tubes last?

Results(Insert your table (with a descriptive title!) below)

Discussion1. Explain your results in terms of your hypothesis.

2. How could you expand this experiment to provide additional evidence?