December 12, 2013Group 6ENZYMES

Rey John D. Caballero1, Jules Carl R. Celebrado1, Charlene Z. Diocancil1, and Kathleen V. Manuel1 1Biology Student, Department of Biology, College of Science, Polytechnic University of the Philippines

ABSTRACT An enzyme is a protein that catalyzes or speed up chemical reactions. The optimum reaction conditions are different for each enzyme. The correct environmental conditions, proper substrates, and, often, particular cofactors associated with an enzyme are needed. Denaturation occurs when it is subjected to excessive heat or extremely high or low pH (denaturing conditions). This laboratory activity will help to develop a concise understanding of a specific enzymatic reaction and to understand more which factors that affect enzyme activity that could be biologically important. To test the activity of amylase, sucrase, hydrolase and catalase various techniques are conducted to show the presence or absence of a material that reacts in the specific enzyme. Enzymatic proteins are fundamental to the survival of any living system and are organized into a number of groups depending on their physiological processes they are involved. Keywords: Enzymes, catalyes, amylase, sucrase, hydrolase, catalaseINTRODUCTIONAn enzyme is a protein that serves as a biological catalyst (Denniston, 2007). A catalyst is any substance that increases the rate of a chemical reaction (by lowering the activation energy of the reaction) (Denniston, 2007).They are large protein molecules, folded so that they have very specifically shaped substrate binding sites. These binding sites make substrates go into the transition state. To catalyze the reaction, several regions of the binding site must be precisely positioned around the substrate molecules. Any change in the shape of the overall folded enzyme molecule can change the shape of the binding site.Enzymes are highly specific for certain reactants; the compound acted upon by the enzyme is known as the substrate. The names of enzymes most commonly end in the suffix -ase, which is sometimes appended to the name of the substrate or the type of reaction. Some enzymes function properly only in the presence of cofactors or coenzymes. Cofactors are inorganic, often metallic, ions such as Mg++and Mn++, while organic molecules such as NAD, NADP, and some vitamins are coenzymes. Both cofactors and coenzymes are loosely associated with enzymes; however, prosthetic groups are nonprotein molecules that are attached to some enzymes and are necessary for enzyme action.Enzymes speed up chemical reactions by lowering activation energy (that is, the energy needed for a reaction to begin). In every chemical reaction, the starting materials (the substrate(s) in the case of enzymes) can take many different paths to form products. For each path, there is an intermediate or transitional product between reactants and final products. The energy needed to start a reaction is the energy required to form that transitional product. Enzymes make it easier for substrates to reach that transitional state. The easier it is to reach that state, the less energy the reaction needs. . The optimum reaction conditions are different for each enzyme. The correct environmental conditions, proper substrates, and, often, particular cofactors associated with an enzyme are needed. Enzyme denaturation occurs when it is subjected to excessive heat or extremely high or low pH (denaturing conditions). When an enzyme is denatured it loses its quaternary, tertiary and secondary structure and becomes a chain of amino acids linked by peptide bonds (or covalent bonds that occur between adjacent amino acids).Enzymatic proteins are fundamental to the survival of any living system and are organized into a number of groups depending on their specific activities. Catalytic enzymes that break down proteins, which are called proteases, are found in many organisms; one example is bromelain, which comes from pineapple and can break down gelatin and is often an ingredient in commercial meat marinades. Anabolic enzymes are equally vital to all living systems. One example is ATP synthase, the enzyme that stores cellular energy in ATP by combining ADP and phosphate. In addition to making life possible, many enzymes have numerous applications that affect our daily lives in other ways such as food processing, clinical diagnoses, sewage treatment, and the textile industry.This laboratory activity will help to develop a concise understanding of a specific enzymatic reaction and to understand more which factors that affect enzyme activity that could be biologically important.METHODOLOGYCorn (Zea mays) seedlings, germinated mungbean (Vigna radiata) sprouts and potato (Solanum tuberosum) used as a botanical material in this experiment.In examining different hydrolases in the plant, two enzymes under hydrolases are observed and experimented (Amylase and Sucrase). In detecting the presence of amylase on Zea mays, two test tubes are prepared and labeled that contained 10mL of 0.1% starch solution. Two freshly detached root systems from the corn seedlings are soaked in the starch solution of test tube 1, and then both test tubes are incubated overnight at room temperature. A drop of I2Kl was added in each tube after incubation (root systems removed). Test tubes are shook and compared according to their color intensities. For sucrase, two test tubes are set up and labeled and then each test tube were added with 5mL of 1.0% sucrose solution. Two freshly detached root systems of corn seedlings are dipped in the sucrose solution of test tube 1, and then both tubes are incubated at room temperature overnight. After incubation, each tube (root systems removed) are added with same volume of Benedicts solution and heated in water bath for testing reducing sugars in the solution. In investigating different oxidoreductases in plants, two (2) enzymes under oxidoreductases are used and experimented (Dehydrogenases and Catalases).In observing dehydrogenases in Vigna radiata, two (2) big test tubes are marked and filled up to its brim with 0.001% of methylene blue. 10g of freshly germinated Vigna radiata (seed coat removed) was then added to test tube 1 and then each test tubes are sealed with a stopper (make sure that the set up have no air bubbles). Both tubes are incubated overnight at room temperature and observed for any change in color with test tube 2 as control.In observing catalases in Solanum tuberosum, two (2) test tubes are labeled and pipetted with 5mL of 3% hydrogen peroxide. Six (6) 2cm thin strips of freshly peeled Solanum tuberosum are prepared; the first three strips are boiled for 3 minutes while the other three are raw. After boiling, the strips are drop at the same time in both tubes (3 potato strips per test tubes),and then observed for gas evolution after 5 to 10 minutes.The results were presented using tables and pictures.RESULTS AND DISCUSSIONSA. Hydrolases

A.1 Amylase

BA

Figure 1 (A-B): Enzyme activity of Amylase; both test tubes contains 0.1% starch solution. In Test tube 1 having a detached corn seedling root (fig. A.1) while test tube 2 served as the control (fig A.2); In figure B. There is a precipitate present in test tube 2

Table 1 Reaction of 0.1% starch solution in with and without detached root of corn seedling after 24 hours

OriginalAfter 24 hours(after adding I2KI)

With corn rootNo reaction takes placeNo purple precipitate forms(negative in starch)

Without corn root No reaction takes placeForms purple precipitate(positive in starch)

After incubating the two test tubes at room temperature overnight, the root systems are removed from the starch solution. In test tube 2, after adding I2KI, purple precipitate form below while in test tube 1 where detached corn root systems was placed showed no product or precipitate form after incubation.I2KI or potassium iodide is used to test for starches. In our observation as we dropped Potassium iodide in the test tubes, the test tube 1 that has a root system has no changes in color, it is just low and blurry and there is no presence of purple particles at the bottom indicates the absence of starch within the set-up. On the test tube 2 that is a control, has a color violet precipitate at the bottom which indicates the presence of starch that is due to the reaction of I2KI. We can infer that the starch in the solution in test tube 1 was broken down into usable sugar by the amylase present in the roots and stored for necessary energy and as food storage. Amylase is present in the roots of the newly germinated corn seedlings. It broke down the starch with the presence of water in the solution by the addition of the hydrogen and hydroxyl ions of water to a molecule with its consequent splitting into simpler sugar molecules which are stored in the roots as food and energy for plant. Test tube 1 was negative in I2KI test because it has no reaction or changes in color that indicates the presence of starch.Amylaseis anenzymethatcatalysesthehydrolysisofstarchintosugars. Amylase in plants assists in the initial development of the plant, before it is able to use energy from photosynthesis. The amylase enzymes begin their role in plant development as the plant's seed begins to germinate, root, and sprout. Asdiastase amylase was the first enzyme to be discovered and isolated byAnselme Payenin 1833. All amylases areglycoside hydrolasesand act on -1,4-glycosidic bonds. Amylase is an enzyme that acts with the presence of water molecules to hydrolyze carbohydrates. The role of amylase in plants is to break down starch molecules. Starches are usually processed in this way during seed germination, and turned into sugar which provides sources of energy for the plant during its early development. Plants are able to store energy from the sun by creating sugar. As baird and Arevalo said, Without the presence of amylase, a seedling would not be able to grow to reach the sunlight needed for photosynthesis and healthy growth.A.2 Sucrase (Invertase)

Figure 2 After Adding Benedicts Solution and Heating

Table 2 Presence of Reducing Sugars and Enzymatic Activity of Sucrase

Test TubeResults

1Green (positive)

2Blue (negative)

Color Range: (None) -----Blue-----Green----Yellow-----Orange-----Red----- (Abundant)

In the experiment, we put the fresh Zea maize roots with 5 ml of 1.0 % sucrose solution in test tube 1. We filled up test tube 2 with sucrose solution only for it would be our control for this activity. After incubating for 24 hours, we removed the roots in test tube 1 then added 5 ml of Benedict's solution on both tubes proceeded by heating. In Test tube1 the resulting color is green while in test tube 2 is blue.The amount of glucose formed is directly proportional to the reaction rate. The more active the sucrase (invertase), the more sucrose will be broken down, and the more glucose will form. This will be indicated by the color change when testing with Benedict. Test tube 2, which remained blue in color, implies that the result is negative, for sucrose is a non-reducing sugar. (fig 2.2). Since test tube 1 reacted and showed a color change from blue to green (fig. 2.1), reducing sugars were present. In testing with Benedicts reagent, there must be a color change of orange-red. Using a color range, it was indicated that test tube 1 contained small amount of reducing sugars since it only change its color partially. Therefore, test tube 1 with Zea maize roots showed minimal enzymatic activity of sucrase because small amount of reducing sugars were present. The enzyme sucrase was not active enough to break down sucrose into glucose and fructose form.Invertase is a key metabolic enzyme which hydrolyzes the disaccharide sucrose (the major type of sugar transported through the phloem of higher plants) to glucose and fructose. In higher plants, invertase exists in several isoforms with different biochemical properties and subcellular locations. The specific functions of the different invertase isoforms are not clear, but they appear to regulate the entry of sucrose into the different utilization pathways. Invertase, alone or in combination with plant hormones, are involved in regulating developmental processes, carbohydrate partitioning, as well as biotic and abiotic interactions.

B. Oxidoreductases B.1 Dehydrogenases

CBA

Figure A-C: Test tubes having a brim-filled of 0.001% Methylene blue; Fig. A with and without Mung bean seedlings before incubation; Fig. B- after incubation within 24 hours and Fig. C-after aeration

Table 3 Reaction of Methylene Blue on with and without Mung bean Seedlings

Test tube with 0.001% methylene blueColor

Original After 24 hoursAfter Aeration

With germinated Mung bean seedlingsBlueColorlessBlue

without germinated Mung bean seedlingsBlueBlueBlue

Two test tubes were brim-filled with 0.001% methylene blue. The first test tube has a Mung bean seedlings and the second test tube without mung bean seedlings served as the control (see figure A). It was covered tightly with a stopper and was incubated overnight. After 24 hours there was a change of color in test tube 1. From blue it becomes colorless and the test tube 2 remains unchanged (see figure B). After aeration, the solution in test tube 1 becomes blue in color again (see figure C).Methylene blue acts as an artificial electron acceptor (oxidizing agent). It is blue when oxidized, but turns colorless when reduced due to the stopping of air to flow inside the test tube. Methylene blue can, therefore, be used to show the presence of active dehydrogenase enzymes by a color change. Dehydrogenase enzymes remove hydrogen from their substrate. As a result, oxygen is liberated and is free for take up of the seedling. Methylene blue is reduced and seed gets its needed oxygen. Presence of dehydrogenase in germinating mung bean seedlings reduced the methylene blue. When methylene blue is substituted for NAD+, the blue color of methylene blue will disappear as it is reduced, thus, change it from blue to colorless. NADH or reduced methylene blue can be oxidized by the mitochondrial respiratory electron transport system when oxygen is available. This will result in the blue color of methylene blue reappearing upon reoxidation by the respiratory chain. Oxidoreductases are a class of enzymes that catalyze oxidoreduction reactions. Oxidoreductases catalyze the transfer of electrons from one molecule (the oxidant) to another molecule (the reductant). Oxidoreductases catalyze reactions similar to the following, A+ B A + Bwhere A is the oxidant and B is the reductant. It can be oxidases or dehydrogenases. Oxidases are enzymes involved when molecular oxygen acts as an acceptor of hydrogen or electrons. Whereas, dehydrogenases are enzymes that oxidize a substrate by transferring hydrogen to an acceptor that is either NAD+/NADP+or a flavin enzyme. Although a great deal of information has been amassed concerning dehydrogenases in animal tissues, there was for a long time little evidence that certain of these important enzymes even existed in plants. Malic and citric dehydrogenases were reported in 1929 in cucumber seeds (Thunberg 1929), but it was not until 1939 that succinic dehydrogenase was found, first in pollen. (Okunuki 1939) and then in certain other tissues (Damoran1941). Nevertheless, the apparent absence or near-absence of succinic dehydrogenase in some tissues (Bartlett 1943) as well as the occasional reports of the presence of individual enzymes (Thunberg 1938) seemed to indicate that the dehydrogenases, at least those of the 4 carbon and 6 carbon acids, were distributed only sporadically. It was during this period that the tricarboxylic acid cycle of Krebs (Krebs 1943), embodying many of these dehydrogenases, was becoming accepted as the main pathway of respiration in animal tissues. Respiration studies in plants (Bonner 1948) pointed in the same direction.

B. 2 Catalases

Figure 4: from L-R, reactions of potatoes between raw and boiled after 5 minutes; reaction of potatoes raw and boiled after 10 minutes

Table 4: Reactions of raw and boiled potato to hydrogen peroxides (H2O2)

Potato stripsObservation

Boiled potato strips No reaction upon contact with hydrogen peroxide (H2O2)

Raw potato stripsProduce bubbles upon contact with hydrogen peroxide (H2O2)

The raw potato strips produce foam (see test tube 1) while the boiled potato strips do not react upon contact with hydrogen peroxide (H2O2) (see test tube 2). The raw potato strips produce foam upon contact with hydrogen peroxide (H2O2), because the catalase in the potato strips react with hydrogen peroxide (H2O2) and change it into water (H2O) and oxygen gas (O2). The bubbles at top are pure oxygen bubbles while water settles below while the boiled did not produce any bubbles upon contact with hydrogen peroxide (H2O2) because boiling the potato denatures the protein enzyme (catalase) in the potato.Catalases are produced inside the cell that is why the potato was cut to strips, to destroy some cells first. Three strips are boiled and the others were not to test the presence of catalase in different temperatures, the rapid production of bubbles in raw shows that the catalase is doing the reaction fast. The cooked potato strips did not produce any bubbles because the structure of the catalase was altered, heating, increasing salinity etc. denatures a protein, once the structure is changed it will not work anymore; therefore no oxygen no bubbles.The process of photorespiration can be explained as when the plants receives too much light and not enough water, that results excessive production of hydrogen peroxide. Hydrogen peroxide (H2O2) is a by-product of respiration and is made in all living cells. Hydrogen peroxide is harmful and must be removed as soon as it is produced in the cell but left to its self hydrogen peroxide will slowly lose the extra oxygen and change into water that is why cells make the enzyme catalase to speed up the reaction and remove hydrogen peroxide (Hopkins and A. Huner, 2008). Catalases are protein enzymes that react with hydrogen peroxides (H2O2) (P. George, 1947), it can be found in animals it is mostly produced by the liver and heart, in plants some catalases helps in the breakdown of the toxic hydrogen peroxide (H2O2) during respiration for the production of glycine to serine during the glycolate cycle (Leegood et al., 2000), while some chloroplastic enzymes helps in reducing hydrogen peroxides (H2O2) to water (Apel and H. Hirt, 2004). Plants use these kinds of chemicals to avoid oxidative damage or changing hydrogen peroxide to a highly toxic hydroxyl radical (OH-) (Demmig-Adams et al., 2006).STUDY QUESTIONS1. Give the: (1) name of enzymes catalyzing the following chemical reactions, (2) their cellular localization, and the (3) plant physiological process involved.A. Pyruvate + NAD+ +CoA Acetyl-CoA + NADH + H+ + CO2

(1) Pyruvate Dehydrogenase catalyzes the reaction(2) The reaction takes place in the mitochondrion.(3) Krebs cycle is the process involved an event in cellular respirationB. Ribulose- 1,5 bisphosphate + CO2 2 (3- phosphoglyceric acid)

(1) The reaction was catalyzed by RuBisCO or Ribulose 1,5-bisphosphate carboxylase (2) It takes place in stromal space of the chloroplast(3) It involves the process of Carbon Dioxide Fixation (as for Photosynthesis/ Calvin Cycle)C. fructose-6-phosphate + ATP fructose-1,6-bisphosphate + ADP(1) Phosphofructokinase (PFK) catalyzes the reaction(2) The reaction takes place in the cytosol of cells(3) Glycolysis is the process involved (an event in cellular respiration)2. Describe the rate of enzyme catalyzed reaction with increasing substrate concentration.It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum. After this point, increases in substrate concentration will not increase the velocity (delta A/delta T). It is theorized that when this maximum velocity had been reached, all available enzyme has been converted to enzyme substrate complex (Freshcorn et. al., 2008). The Michaelis constant Km is defined as the substrate concentration at 1/2 the maximum velocity. Using this constant and the fact that Km can also be defined as: Km=K-1 + K2 / K+1. Michaelis constants have been determined for many of the commonly used enzymes. The size of Km tells us several things about a particular enzyme (Freshcorn et. al., 2008).a. A small Km indicates that the enzyme requires only a small amount of substrate to become saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations.b. A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity.c. The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently assumed to be enzyme's natural substrate, though this is not true for all enzymes.

A simple chemical reaction with a single substrate shows a linear relationship between the rate of formation of product and the concentration of substrate

Figure 4.1. Rate of Enzymatic reaction based on substrate concentration (Linear)For an enzyme catalization reaction, there is usually a hyperbolic relationship between the rate of reaction and the concentration of substrate.

Figure 4.2 Rate of Enzymatic reaction based on substrate concentration (Hyperbolic)At low concentration of substrate, there is a steep increase in the rate of reaction with increasing substrate concentration. The catalytic site of the enzyme is empty, waiting for substrate to bind, for much of the time, and the rate at which product can be formed is limited by the concentration of substrate which is available. As the concentration of substrate increases, the enzyme becomes saturated with substrate. As soon as the catalytic site is empty, more substrate is available to bind and undergo reaction. The rate of formation of product now depends on the activity of the enzyme itself, and adding more substrate will not affect the rate of the reaction to any significant effect (Freshcorn et. al., 2008).

Figure 4. 3. Limitation of substrate concentration in an enzymatic reactionThe rate of reaction when the enzyme is saturated with substrate is the maximum rate of reaction, V max. The relationship between rate of reaction and concentration of substrate depends on the affinity of the enzyme for its substrate. This is usually expressed as the Km (Michaelis constant) of the enzyme, an inverse measure of affinity. For practical purposes, Km is the concentration of substrate which permits the enzyme to achieve half V max. An enzyme with a high Km has a low affinity for its substrate, and requires a greater concentration of substrate to achieve V max (Freshcorn et. al., 2008). 4. In what ways does hydrogen ion concentration affect enzyme activity?The pH scale is the logarithm of the reciprocal of hydrogen-ion concentration in gram atoms per liter; provides a measure on a scale from 0 to 14 of the acidity or alkalinity of a solution (where 7 is neutral and greater than 7 is basic while lesser than 7 is acidic). Hydrogen ion concentration affects enzyme activity by its relationship to pH. Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate can bind or cannot bind to the active site or it cannot undergo catalysis. High hydrogen ion content caused the breaking of the ionic bonds that hold the tertiary structure of the enzyme in place. The enzyme lost its functional shape, particularly the shape of the active site, such that the substrate no longer fit into it, the enzyme is denatured. The ions also affected the charges on the amino acids within the active site such that the enzyme was unable to form an enzyme-substrate complex (Krause et. al, 1998). In general enzymes have a pH optimum. However the optimum is not the same for each enzyme. CONCLUSIONS

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