Enzyme

EDVOTEK P.O.
Box 1232 West Bethesda, MD 20827-1232
The Biotechnology Education Company
282
EDVO-Kit #
Principles of Enzyme Catalysis

Storage:
See page 2 for specific storage instructions.
Experiment Objective: The purpose of this experiment is to understand enzyme catalysis. Students will perform an enzyme assay and determine the rate of a biochemical reaction.
1-800-EDVOTEK (301) 251-5990 24-hour FAX: (301) 340-0582 http://www.edvotek.com email: edvotek@aol.com
All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals.
EVT 910148AM
EDVO-Kit # 282: Principles of Enzyme Catalysis
EDVOTEK The Biotechnology Education Company
Major Section Headings

Page 2 3 4 9 12 13
None of the experiment components have been prepared from human sources.
Experiment Components Requirements Background Information Experimental Procedures Analysis Study Questions Instructor's Guide Pre-Lab Preparations Expected Results Answers to Study Questions Material Safety Data Sheets
All components of this experiment are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals.
15 20 21 22
Experiment Components
This experiment is designed for 10 groups.
Storage: Store component A in the freezer. All other components can be stored in the refrigerator.
Contents A. B. C. D. Catalase solution 1.2% hydrogen peroxide, stabilized Phosphate buffer, pH 7.2, 20x concentrate Assay reagent, potassium iodide, concentrate E. Acidification solution, 0.1 M HCl F. Color enhancer, concentrate G. Color developer, concentrate One 15 ml plastic tube
Storage Freezer Refrigerator Refrigerator Refrigerator Refrigerator Refrigerator Refrigerator
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1990,1991,1997, 1998, EDVOTEK, Inc., all rights reserved. EVT 910148AM
Requirements
Visible wavelength spectrophotometer Test tube racks Timers or clock with second hand Lab permanent markers Test tubes (13 x 100 mm, 10 ml) Beakers Distilled water 5 and 10 ml pipets Pipet pumps or bulbs Linear graph paper 20 - 1 ml pipets, reusable glass or plastic with 0.1 ml divisions, or an automatic pipetor (200-1000 l) and tips (more will be required for Instructor Pre-Lab) Ice
BACKGROUND INFORMATION
Principles of Enzyme Catalysis

A catalyst is a substance that accelerates the rate of a chemical reaction without being consumed or transformed by the reaction. The equilibrium constant of the reaction is not altered by the catalyst. Only the rate of approach to equilibrium is changed. A catalyst is not required in stoichiometric quantities and is often used in trace amounts. Platinum, palladium, strong acids, and bases are frequently used catalysts in organic chemistry and can accelerate reactions thousands of times. The great majority of chemical reactions in the cell are catalyzed by the biological catalysts known as enzymes. Enzymes can accelerate reactions 1014 to 1020 times, amounts which are far greater than any artificial catalyst. Enzymes generally produce these accelerations under the comparatively mild physiological conditions of neutral pH, atmospheric pressure and temperatures of 37C. Unlike most catalysts, enzymes are generally very specific for the reactions they catalyze. The activity of certain enzymes can also be regulated by intracellular concentrations of key metabolites not directly involved with the reaction. This regulation can increase or decrease the activity of the enzyme in a manner adaptive to the cells physiological requirements at a given time. Enzymes that are regulated in this way are termed allosteric. In 1897 Eduord Buchner demonstrated that cell free extracts from yeast could catalyze alcoholic fermentation of sugar. In 1926 J.B. Sumner demonstrated that an enzyme was a protein. With the exception of specialized RNA molecules involved in RNA self-splicing and certain cyclodextrins, all naturally occurring bio-catalysts to date are proteins. Thousands of different enzyme activities are known. Proteins are physico-chemically very diverse and complex. However, there are certain common features to structures and function. Proteins consist of specific sequences of amino acid residues linked to each other by peptide bonds. The sequence of residues in the polypeptide chain is called primary structure. The chemical variety of the amino acid functional groups (e.g., hydroxyl, carboxylic acid, amino, guanido, phenolic, sulfhydryl) are largely responsible for the chemical activity, binding specificities, and electrical properties of proteins. The nonpolar hydrocarbon groups are important in maintaining the overall structure of protein and creating the appropriate chemical environments within the protein. The backbone of the polypeptide chain consists of peptide bonds. The folding path of the backbone through space is called secondary struc-
Background Information, continued
ture. The patterns are complex, having bends, twists and spirals. Secondary structure is mainly determined by hydrogen bonds between backbone oxygens, nitrogens and hydrogens. Well known examples of secondary structure include -helices and -pleated sheets. Secondary structure is influenced by the type of amino acids present in that part of the polypeptide chain. The complete three dimensional folding pattern of a polypeptide chain, including all the positions of the amino acid functional groups is called the tertiary structure. The tertiary structure creates the crevices and pockets which enable the protein to bind and react with other molecules. It also gives the protein its shape and affects its solubility. Most importantly, the precise tertiary structure is absolutely necessary for the proteins biological activity. Many proteins consist of several polypeptide chains that are specifically associated with each other by non-covalent and covalent bonds. The three dimensional arrangement of polypeptide chains to each other in a protein is called quaternary structure. The individual polypeptide chains that make up the protein are often called subunits. The subunits of a protein can be identical, similar, or completely different from one another. Different subunits can be responsible for different functions within a protein. Certain proteins also contain, as integral parts of their structure, chemical groups that are not part of the amino acid residues but are absolutely required for biological activity. These groups include small organic molecules, such as certain vitamin derivatives, and certain metal ions. Moieties such as these are called prosthetic groups. A well known prosthetic group is heme. Heme consists of an iron atom coordinated to the nitrogens of a set of organic rings called porphyrin. A protein that contains all its natural structural elements and possesses biological activity is called native. When a protein has been unfolded, it no longer possesses biological activity even though the backbone and the amino acid groups remain intact. Unfolding also causes subunit dissociation if there are no intersubunit covalent links between them. Unfolded, inactive proteins are called denatured. The reactant molecule in an enzyme catalyzed reaction is called the substrate. The substrate (S) is transformed to product (P). Before the enzyme can transform the substrate it must first bind to it. Initial binding is non-covalent and can be in rapid equilibrium. After productive binding has been achieved, the enzyme-substrate complex can now generate product which is subsequently released. The free enzyme (E) can now react with more molecules of substrate (the enzyme has turned

over). This can be summarized using a single substrate, single product, non-reversible reaction: E+S
ES
EP
E+P
The disappearance of S or the appearance of P (or both) can be measured as a function of time. This relationship is the rate of the reaction. The method of measurement is called the assay. At a fixed enzyme concentration and fixed reaction conditions, the reaction rate can be increased by increasing substrate concentrations. The probability of forming more ES complex increases when there are more substrate molecules present. Generally, the substrate concentration is thousands of times greater than the enzyme concentration in kinetic studies performed invitro. At the early stages of the reaction, if the substrate concentration is in great excess, the rate is approximately linear with time and is termed the initial velocity (v). [S]1 - [S]2 ______________ T1 - T2 where [S]1 is the molar concentration of substrate at some initial time, T1, and [S]2 is the substrate concentration at a later time, T2. The reaction rate can also be expressed in terms of the appearance of product: [P]2 - [P]1 ________________ T 2 - T1 Note that the concentration of substrate decreases with time and the concentration of product increases with time. Graphically, this can be represented with the substrate concentration on the y-axis and time on the x-axis. The decrease in the substrate concentration with time will generate a curve. The rate of decrease is fastest at the earliest times of the reaction since the substrate concentration is comparatively high. The rate of decrease diminishes at later times because the substrate concentration is lower and the reaction is slower. Within small time intervals there will be sections of the curve that are approximately linear and the rate can be estimated. The rate of an enzyme reaction cannot be increased indefinitely by continuously increasing the substrate concentration. At some substrate concentration, all the enzyme molecules are bound to substrate and are involved in some stage of the catalytic cycle. Under these conditions the enzyme is saturated with substrate and no further increase in reaction velocity is observed.

A great deal of information can be learned about enzyme mechanisms by studying reaction rates. Reaction rates are frequently used in clinical chemistry to assess the blood levels of various enzymes in disease. Only a relatively small portion of the enzyme molecule is involved with substrate binding and catalysis. This region is called the active site. The active site contains the critical amino acid residues and, if applicable, the prosthetic groups required for activity. The catalytic site of enzymes is an active area of research since the various physical and chemical mechanisms believed to be responsible for catalysis and substrate recognition occur there. Any agents or conditions that denature an enzyme will destroy its activity. Denaturation can be caused by heat (temperatures greater than 45C, but there are exceptions), extremes in pH, organic solvents, and cycles of freezing and thawing. Ionic detergents, such as sodium dodecyl sulfate, are potent protein denaturants. Other agents disrupt protein structure by direct chemical reaction with the amino acid residues. Examples include heavy metals, free radicals and peroxides. Hydrogen peroxide is a toxic by-product of aerobic oxidation and certain processes in intermediary metabolism. All aerobic life forms have evolved methods of enzymatic peroxide detoxification. The enzyme catalase (H2O2:H2O2 oxidoreductase) catalyzes the rapid decomposition of hydrogen peroxide by the following reaction: 2 H2O2 > 2H2O + O2 (gas) Catalase uses the hydrogen peroxide as a hydrogen acceptor and donor. This activity is called catalatic. The enzyme can also utilize short chain organic peroxides. Other hydrogen donors, such as ethanol, phenols and formate, can be used by the enzyme to reduce the hydrogen peroxide. This activity is called peroxidative. The catalatic activity is preferred in-vitro. Almost all the cell types in mammals contain catalase, with liver, kidney and erythrocytes being particularly rich sources. Bovine liver catalase has a molecular weight of approximately 250,000 and is a tetramer of 4 identical subunits. The enzyme contains 4 heme prosthetic groups per molecule, one per subunit. The heme forms part of the active site and there are 4 active sites per molecule of enzyme. The peroxide oxygens are believed to be coordinated to the heme iron during one phase of the catalytic cycle. Another molecule of peroxide is then used to complete the reaction. Catalase has one of the highest turn over rates known. Over 3.6 x 107 molecules of hydrogen peroxide are decomposed per minute per molecule of enzyme.

There are several methods that can be used to assay for catalase activity. The most direct assay involves monitoring the hydrogen peroxide concentration by ultra-violet spectrophotometry in-situ at 240 nanometers. A method frequently used involves titration of peroxide with potassium permanganate in high concentrations of sulfuric acid. This method uses hazardous chemicals, is somewhat cumbersome and time consuming. EDVOTEK has developed a colorimetric assay to measure the concentration of hydrogen peroxide based on the oxidation of iodide by peroxide: 2 I- + 2 H+ + H2O2 > 2 H2O + I2 The iodine imparts a brown-red color to the solution. The color intensity increases with the peroxide concentration. Consequently, catalase mediated decomposition of hydrogen peroxide will decrease the color intensity of the assay with time. The stoichiometry of this reaction creates 1 mole of iodine for every mole of hydrogen peroxide. The assay has been designed to use a minimum of acid (less than 10 mM HCl). For a typical catalase assay the iodine concentration generated is less than 0.2 mM.
EXPERIMENTAL PROCEDURES
EXPERIMENT OBJECTIVE:
The purpose of this experiment is to understand enzyme catalysis. Students will perform an enzyme assay and determine the rate of a biochemical reaction.
LABORATORY SAFETY
Gloves and goggles should be worn routinely as good laboratory practice.
Student Experimental Procedures

The enzyme catalase catalyzes the decomposition of hydrogen peroxide (substrate) to water and oxygen gas (products). In this experiment:
Remember!
Catalase will be added to a buffered solution of hydrogen peroxide. A time course of the reaction will be obtained by removing aliquots from the reaction tube every 30 seconds. These aliquots will be added to separate tubes of assay solution. The assay solution denatures the enzyme, catalase, which destroys its activity. The iodide (I-) in the assay solution is oxidized by any remaining peroxide, producing a red-brown iodine ( I2 ) solution. The color intensity can be quantitated in the spectrophotometer and the rate of the reaction determined. The concentrations of peroxide and enzyme in the reaction are approximately 1.8 milliMolar and 5 nanoMolar respectively.
Wear gloves and safety glasses. Label your tubes at the top with a permanent marker.
PREPARATION OF ASSAY TUBES

1. With a water resistant pen, label 6 empty test tubes (at the top) to indicate blank and the various reaction times (0 to 2 minutes): B (blank) 0 0.5 1.0 1.5 2.0
10
Student Experimental Procedures, continued

2. With a 5 ml pipet, transfer 3 ml of assay solution to each of the tubes. Rinse pipet liberally with distilled water (about 200 ml). Save for Step 5. With a 1 ml pipet, transfer 0.3 ml of diluted buffer to the blank. Mix by tapping or vortex. Save pipet and use in Step 6.
3.
WEAR SAFETY GOGGLES AND GLOVES
PREPARATION OF CONTROL AND REACTION TUBES

4. Label one of two remaining test tubes "Con" (for control) and the other "Rxn" (for reaction). With the rinsed 5 ml pipet, dispense 1.8 ml of Enzyme Reaction Cocktail to each of the two tubes. With a 1 ml pipet, add 0.3 ml of dilute phosphate buffer to the control tube. Save the pipet for Step 7. Remove 0.3 ml of liquid from the control tube and add to the assay tube labeled 0. Set the "Con" tube aside and continue with the "Rxn" tube. Discard pipet.
Remember!
5.
6.
Upon addition of enzyme catalase, the reaction will begin. Start timing the reaction immediately and aliquot 0.3 ml of this mixture to tubes labeled 0.5, 1.0, 1.5 and 2.0 at 30 second intervals.
7.
Enzyme reaction cocktail is added. Hydrogen peroxide that is not catalyzed by the enzyme catalase will oxidize iodide to give a brown-red color. The color intensity increases with the peroxide concentration.
Con Peroxide/Buffer No Catalase Rxn Peroxide/Buffer Catalase
Blank Assay Buffer Phosphate Buffer (used to blank B spectrophotometer for background)
Control Establishes Zero Timepoint 0 0.5
Rxn Tube 1.0 1.5
Catalase (Enzyme) Converts Peroxide into Products Timepoints 0.5, 1.0, 1.5 and 2.0 2.0
Assay Tubes
11
Student Experimental Procedures, continued Preparation and Monitoring of Timed Reactions

8. With a FRESH 1 ml pipet, add 0.3 ml of diluted catalase (Enzyme) to the tube you labeled Rxn. Mix. Start timer, or note the second hand on the clock or watch. Retain pipet to take samples. Rinse pipet liberally with distilled water (about 200 ml) after adding catalase. With the 1 ml pipet, remove 0.3 ml from the Rxn tube and at 0.5 min. (30 seconds), add it to tube 0.5. Mix.
9.
10. With the 1 ml pipet, remove 0.3 ml from the Rxn tube and at 1 minute, add it to tube 1. Mix. 11. With 1 ml pipet, remove 0.3 ml from the Rxn tube and at 1 minute 30 seconds, add it to tube 1.5. Mix. 12. With 1 ml pipet, remove 0.3 ml from the Rxn tube and at 2 minutes, add it to tube 2.0. Mix. Set pipet aside. 13. Wait 4 minutes after your last time point to allow full color development.
Data Collection
Spectral readings can now be taken. Depending on the spectrophotometer, you may be able to insert your test tubes directly into the instrument. Otherwise transfer the entire enzyme reactions in tubes provided by your instructor starting with 0 to 2.0.
TIME (min)
Blank 0 0.5 1.0 1.5 2.0
Assay Solution
3 ml 3 ml 3 ml 3 ml 3 ml 3 ml
Diluted Buffer
0.3 ml -----------
Volume Con
--0.3 ml ---------
Volume Rxn
----0.3 ml 0.3 ml 0.3 ml 0.3 ml
A500
12
Student Experimental Procedures, continued

15. Zero the instrument with the tube B solution (Blank) according to your instructors directions. Be sure the instrument is set at 500 nm wavelength. The instrument should read 0 absorbance with the blank solution (no color). 16. Remove the blank and record the absorbancy values for each solution in tubes 0 to 2.0. Record the results in the table. If you are using an instrument such as a Spec 20, it should take approximately two to three minutes to complete your readings.
Analysis
The reaction rate can be obtained by graphing the absorbancy data versus time. However, the rate can also be expressed in terms of substrate consumed. 1. To express your data in terms of molar concentration of peroxide:
Absorbance x 11 = Molarity of hydrogen peroxide in Rxn tube. e
e is the extinction coefficient for this assay system and has been determined by your instructor and will be provided. Multiplication by 11 (dilution factor) gives the peroxide concentration in the reaction tube. Scientific notation will make the calculations more convenient. 2. Graph the peroxide concentration on the y-axis versus time on the x-axis. Draw the best straight line through the data points. You may notice some curvature to the data points. This is normal, especially between 0 and the first time point, and between later time points. You are making a linear approximation. Determine the rate of change in the molarity of hydrogen peroxide with time. The rate is equivalent to the slope of the line. Pick a time, go vertically up to the line, then horizontally to the y-axis. Determine the concentration in this way for the next time point. rate = [peroxide]1 - [peroxide]2 | time 1 time 2 |
3.
4.
Express the rate as molarity change per minute.
13
Study Questions
1. Why did you observe bubbles in Step 18 of the experiment? Assume you had boiled the enzyme solution before adding it to the peroxide. Would you expect to see bubbles? What gas do the bubbles contain? Why did the color intensity of your peroxide assays decrease with time? What makes the rate of a reaction of an enzymatic reaction decrease? Assuming optimal reaction conditions (pH, temperature, etc.) how could you increase the rate of the reaction other than increasing the substrate concentration? An active preparation of catalase was exposed to the proteolytic enzyme, trypsin. The catalase preparation was found to be inactive when it was reassayed. Why? Concentrated solutions of catalase have a red color. Why? The velocity of a catalase reaction was found to increase with increasing hydrogen peroxide concentrations as expected. However, at high peroxide concentrations, the reaction rate decreased and eventually went to zero. What could explain this observation? Which of the following generalized enzyme- catalyzed reaction schemes best describes the catalase reaction? a. E + S
2.
3.
4.
5.
6. 7.
8.
ES EP
ES1S2
E + P E + P
b. E + S1 + S2 c. E + S
EP
ES EP1P2 E + P1 + P2
ES1S2
d. E + S1 + S2
EP1P2 E + P1 + P2
14
Notes:

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EDVOTEK P.O.

Box 1232 West Bethesda, MD 20827-1232

The Biotechnology Education Company

Principles of Enzyme Catalysis

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

Major Section Headings

Storage Freezer Refrigerator Refrigerator Refrigerator Refrigerator Refrigerator Refrigerator

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

Principles of Enzyme Catalysis

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

Background Information, continued

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

Background Information, continued

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

Background Information, continued

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

Background Information, continued

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

Student Experimental Procedures

PREPARATION OF ASSAY TUBES

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

Student Experimental Procedures, continued

WEAR SAFETY GOGGLES AND GLOVES

PREPARATION OF CONTROL AND REACTION TUBES

Control Establishes Zero Timepoint 0 0.5

Rxn Tube 1.0 1.5

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

Student Experimental Procedures, continued Preparation and Monitoring of Timed Reactions

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

Student Experimental Procedures, continued

Express the rate as molarity change per minute.

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

EDVO-Kit # 282: Principles of Enzyme Catalysis

EDVOTEK The Biotechnology Education Company

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