...Chapter 3 Enzymes Learning Outcomes Candidates should be able to: (a) explain that enzymes are globular proteins that catalyse metabolic reactions; (b) explain the mode of action of enzymes in terms of an active site, enzyme/substrate complex, lowering of activation energy and enzyme specificity; (c) [PA] follow the progress of an enzyme-catalysed reaction by measuring rates of formation of products (for example, using catalase) or rates of disappearance of substrate (for example, using amylase); (d) [PA] investigate and explain the effects of temperature, pH, enzyme concentration and substrate concentration on the rate of enzymecatalysed reactions; (e) explain the effects of competitive and non-competitive inhibitors on the rate of enzyme activity; (f) use the knowledge gained in this section in new situations or to solve related problems. Enzymes are globular protein which act as catalysts • Enzymes are protein molecules defined as biological catalysts which speed up a chemical reaction and remain unchanged at the end of reaction. • Enzyme names end in –ase E.g. amylase, ATPase • Enzyme are globular proteins. Enzymes are globular protein which act as catalysts • Enzyme molecules are coiled into a precise threedimensional shape, which hydrophilic R groups (side-chains) on the outside of the molecule ensuring that they are soluble. Enzymes are globular protein which act as catalysts • Enzyme molecules have active site. • The active site of an enzyme is a region...
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...Effects of Temperature, pH, Enzyme Concentration , and Substrate Concentration on Catecholase Introduction Enzymes are biological proteins that speed up the reaction rate of a chemical reaction. They work in the human body by lowering activation energy making certain that reactions will initiate. For every action, there is an equal and opposite reaction. In this case, factors that influence the activity of an enzyme are called modulators. If modulators activate enzymes the reaction rate catalyzed will significantly increase, but if the modulator inactivates enzymes the reaction rate catalyzed will significantly decreased (Silverthorn, 2004). The potentially disastrous influence of temperature, pH, enzyme concentration, and substrate concentration on enzymes and other proteins is one reason why these modulators are very strictly regulated by the body (Silverthorn, 2004). Temperature, a measure of the intensity of heat, is an important factor in the activity of enzymes. The velocity of an enzymatic reaction is influenced by temperature. This is because substrates collide with active sites frequently in the presence of rapidly moving molecules. In addition, although these molecules do move rapidly the speed of the reaction drops sharply. In short, thermal agitation causes protein molecules (enzymes) to denature ( breakdown of protein structures). All enzymes have an optimal temperature at which reaction rates go fastest without denaturing the enzyme (Campbell and Reece, 2002) pH...
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...Introduction (5 marks) Enzymes are globular shaped proteins that are found throughout the body, with their main function being to act as biological catalysts. An enzyme can act to speed up or regulate the rate of the reaction, in order to maintain an efficient rate of biological reactions. Enzymes, whilst having an important role in the reaction of many chemicals within the body, are not consumed in the reaction, and so are able to catalyze many reactions in their life cycle. Enzymes are able to reduce the activation energy of the reaction; the energy required to break bonds between the reactants, and form new bonds in the products, which allows more product to be formed. (Marieb and Hoehn, 2010, pp.51-53). Enzyme activity is affected by changes in the pH of their solution. For each individual enzyme, there is a corresponding pH at which, that particular enzyme’s activity will be at it’s maximum. This is known as the optimum pH. If the pH of the solution is getting closer to it’s optimum pH for that particular enzyme, then the activity of the enzyme, and therefore it’s rate of reaction, will increase. At extremes of pH (either extremely acidic or basic) enzymes tend to become denatured; a state in which they lose all of their biological activity. (Worthington Biochemical Corporation, 2011). Temperature has the ability to increase the reaction rate of chemical reactions, by increasing the kinetic energy of the molecules themselves. By increasing the kinetic energy...
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...Introduction & Hypothesis: Enzymes are important for every living organism, because they are the reason that reactions occur. Although most reactions would take place without enzymes, enzymes allow these chemical reactions to happen at a much faster rate, therefore making cells more efficient (Reece, 2011). Enzymes are catalysts, and almost always proteins, that speed up the rates of reactions by lowering the activation energy without being consumed in the reaction (Helms, 1998). Throughout this experiment, four procedures will be performed to indicate the factors that alter the functioning of enzymes, and the importance that these factors are to be in correct levels in order for a cell to function properly. Proteins are macromolecules with unique polypeptide chains that make up their structure (Reece, 2011). The functions of proteins are dependent on their structure, and since enzymes are almost always proteins, the structure of the enzyme is very important for the enzyme to function. However, there are factors that can disrupt the structures of enzymes. These can be environmental factors such as temperature and pH, or they can be concentration changes, such as an increase or decrease in enzyme or substrate concentration (Eed, 2013). Temperature is an environmental factor that can alter enzyme activity (Reece, 2011). An increase in the kinetic energy of a solution results in an increase in temperature (Reece, 2011). As the temperature increases, the molecules in the...
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...OF ENZYMES Enzymes are extremely important and without them, the reactions in living organisms would be so slow they would hardly proceed at all. They enable metabolic reactions to proceed rapidly at low temperatures, and as well as speeding up reactions they also control them. There are two main groups of enzymes: intracellular and extracellular. INTRACELLULAR: Occur inside cells where they speed up and control metabolism. EXTRACELLULAR: Produced by cells but achieve their effects outside the cell – includes digestive enzymes that break down food in the gut. Each enzyme is usually specific to particular reactions, and are pH sensitive, with every enzyme having its own range of pH in which it functions best. They are not destroyed by the reactions which they catalyse, meaning they can repeat a reaction over and over. (McMonagle,2015) In an enzyme controlled reaction, the substrate molecules bind with the enzyme to form an enzyme-substrate complex. The reaction then takes place and the product leaves the enzyme. As mentioned above, the enzyme (unchanged by reaction) can then be used again. Below is the equation, with the double arrow meaning that the reaction can go either way, depending on the amount of substrates and products – this ensures a equilibrium, meaning the enzyme will switch if there is an abundance of product and not enough substrates, and vice versa. (Advanced Biology, pg124, 2000) ENZYME + SUBSTRATE ENZYME-SUBSTRATE...
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...Lab Report #2 Name: Lab: #9 Enzymes – Experiment #4 Due date: Purpose The purpose of the experiment is to compare and examine the effect of substrate concentration on catalase activity. Introduction All chemical reactions require a catalyst. A type of catalyst that exists is an enzyme, which acts to bring out a specific biochemical reaction. At all times, all work inside a cell is being performed by enzymes (Brian, 2000). The purpose of an enzyme is to help the cell carry out reactions very quickly. An interaction must be made for a reaction to become catalyzed. The active site is where this interaction between the enzyme and the reactant and/or reactants takes place. In order for the enzyme to work efficiently and properly, the reactant (or substrate) must position itself perfectly within the active site. Most enzymes usually only can catalyze a single chemical reaction, which is called specificity (Introduction To Enzymes, n.d.). Enzymes can also operate to an optimal extent where chemical reactions can occur rapidly and with the upmost efficiency, under certain conditions known as the enzyme’s optimum activity (Boli, 2012). The many different conditions include environmental, such as pH and temperature, or concentrations of the substrates and enzymes. In this experiment, we examined a substance called catalase. Catalase is the isolated cells from potatoes and beef liver. As the substrate for the experiment, hydrogen peroxide was used at various different amounts...
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...Enzymes activity plays an important role on living organisms. Its activity can be affected by different factors, including environmental and molecular elements. Enzyme inhibition by some molecules can be competitive or noncompetitive according to the binding mechanism. The aim of this study was to determine the type of inhibition occurred by the phenylthiourea (PTU) on the catechol oxidase enzyme. Different substrate concentrations, 0.5, 1, and 1.5 mL, and presence (1mL) and absent of PTU was analyzed. Products were measured at 380 nm. Results showed product only when PTU was not present, except for in one tube. A PTU noncompetitive inhibition is proposed based on the inability to obtained product even when the substrate concentration is increased....
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...effect of temperature on Enzyme activity? Purpose: Enzymes are proteins that speed up chemical reactions in cells. They break down molecules called substrates. Each enzymes have only one substrate that breaks down. Enzymes are produced in the cells of the body and affect the rate of almost all the chemical reactions which take place in living organisms. The rate of enzymes activity is influenced by temperature, pH, and substrate concentration. The purpose of this lab was to determine the affects of enzyme activity under specific temperature changes, pH values and substrate concentration. Since heat increases the rate of most chemical reactions, the addition of heat causes faster molecular movement. Most enzymes active in living tissue becomes denatured, their secondary or tertiary protein structure breaks down, at the temperature above 40 degrees C. In the effects of pH, it is expected that the changes in pH would have an effect on the action of enzymes. Lemon juice helps keep the apple from growing, because its full of ascorbic acid (Vitamin C) and it has a low acidic pH level. But extreme high levels of low pH values can result in a complete loss of enzymes activity thus leaving the apple to brown. The effects of substrate concentration, an enzyme substrate complex is formed when a substrate fits into active of an enzyme. The velocity, the rate of speed, at which the enzymes works will increase until it reaches a maximum. If the substrate is present in excess amounts...
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...Effects on Enzymatic Activity Tyler Mitchell BSC 118-002 Shon Johnson November 1, 2011 Abstract Without the effects of enzymes, many crucial chemical reactions would not be able to take place at the rate of which they were meant to perform. We conducted this series of labs in order to discover the effects of different biological and environmental aspects on enzymatic activity. In our first experiment, we looked at the effects that enzyme concentration had on amylase activity. We hypothesized that the higher the concentration of the enzyme, the higher the rate of reaction would take place. We found that a higher concentration of amylase led to a faster rate of reaction, proving our hypothesis to be correct. In our second experiment, we tested the effect the concentration of substrate would have on enzymatic activity. We hypothesized that the lower concentration of the substrate would not change the speed of the reaction. The experiment proved this hypothesis to be wrong because the speed of the reaction slowed down as the concentration lowered. In the third experiment, we tested the effects that different pH levels would have on enzymatic activity. We hypothesized that the more acidic the solution was, the lower the rate of the reaction will be. In Areekijseree’s article, it states that optimal pH levels for amylase are between 4 and 5, and 6 and 8 (Areekisjseree, Engkagul, Kovitvadhi, Thongpan, Mingmuang, Pakkong, Rungruangsak-Torrissen, 2004). This contradicts...
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...BIOL 3380 Name:_____________________________________ Circle Session: T-PM W-AM W-PM R-AM R-PM F-AM F-PM Experiment 9 – Pre-lab Homework Enzyme Kinetics of LDH This pre-lab homework assignment is due at the beginning of your lab session. You are provided with the following portion of a protocol: • Determine concentration of enzyme stock solution, if unknown, by taking an A280 nm reading of a 1:100 dilution (in water). Use a total volume of 1 ml in the cuvette. • Dilute some of the enzyme stock with buffer A to make a 4 mg/ml solution. • Serially dilute the 4 mg/ml solution with buffer A to make working solutions of 400 µg/ml and 40 µg/ml. • Prepare 30 µl of each working solution for every sample The PI of the lab gives you a tube of enzyme and tells you the following before disappearing into the office to write more grant proposals: ➢ There is 50 µl of enzyme stock solution. The enzyme is expensive to purify, so follow the protocol exactly, using as little of the stock solution as possible. ➢ The concentration of the stock solution is currently not known, but a 1 mg/ml concentration of the pure enzyme has an A280 nm of 2.0. ➢ You’ll be performing the assay on 12 samples. ➢ Make enough of each working solution so that you have at least 400 ul to work with when you do the assay (to cover any waste and/or inefficiencies in pippetting). Using the spectrophotometer to read the absorbance at 280 nm, you get...
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...102/103 Lab 4: Enzymes Experiment 1: Effect of Enzyme Concentration Table 1: Effect of Enzyme Concentration on the Production of Gas Tube | Amount of yeast | Balloon circumference (cm) After 1 minute | Balloon circumference (cm) After 3 minutes | Balloon circumference (cm) After 5 minutes | Final Circumference (cm) | Time Required to Complete | 1 | 0.05 g | 15.5 cm | 17.2 cm | 20.5 cm | 21.9 cm | 11 mins | 2 | 0.005 g | 13.8 cm | 15.1 cm | 19.6 cm | 20.1 cm | 12 mins | 3 | 0.0005 g | 8.8 cm | 10.5 cm | 13.1 cm | 13.1 cm | 9 mins | Questions 1. What is the enzyme in this experiment? What is the substrate? Yeast is the enzyme. Sugar as the substrate. 2. Did you notice a difference in the rate of reaction in the tubes with different concentrations of enzymes? Why or why not? It is yes, since there is change in balloon size. More the concentration of the enzyme more will be the consumption of substrate. That indicates the rate of reaction is more. 3. What was the effect of using less enzyme on your experiment? There is less size of the balloon for less enzyme concentration. There is less production in the gas. 4. Do you expect more enzyme activity if the substrate concentration is increased or decreased? Draw a graph to illustrate this relationship. If you increase the substrate concentration the rate will also increase up to a certain point. Where after it will remain the same even with further substrate increases...
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...online: 28 September, 2015 Keywords: Beta: galactosidase, ONPG, X: gal, ONPG and X: gal Corresponding Author: Durve A.* Associate Professor Email: annikadurve ( at ) yahoo ( dot ) com Bhattiwala H. Lecturer Jagtap P. Scientific Officer Abstract In the present study microorganisms producing betagalactosidase enzyme were isolated from fermented millets mixture. Three bacterial isolate C2, C4, Y were found to be Gram positive bacilli, Gram negative cocobacili, Gram positive bacilli respectively while isolate SC2 was found to be yeast. These bacterial isolates were identified using various biochemical tests Growth study kinetics of microbial isolates was performed and the optimum pH and temperature was found. Beta-galactosidase enzyme was extracted and the effect of pH, temperature, substrate variation and incubation period on enzyme activity was studied. The activity of beta-galactosidase for all the microbial isolate increased with increase in temperature upto 37°C. Michaelis-Menten plot and Lineweaver Burk plot were constructed to calculate the Km and Vmax values. Immobilization of crude enzyme from the isolates was performed and enzyme activity using ONPG and lactose as a substrate was found. SDS PAGE analysis of the dialysate sample of bacterial isolate C2 and yeast isolate SC2 was...
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...pH, and substrate concentration on Enzyme activity? Purpose: Enzymes are proteins that speed up chemical reactions in cells. They break down molecules called substrates. Each enzymes have only one substrate that breaks down. Enzymes are produced in the cells of the body and affect the rate of almost all the chemical reactions which take place in living organisms. The rate of enzymes activity is influenced by temperature, pH, and substrate concentration. The purpose of this lab was to determine the affects of enzyme activity under specific temperature changes, pH values and substrate concentration. Since heat increases the rate of most chemical reactions, the addition of heat causes faster molecular movement. Most enzymes active in living tissue becomes denatured, their secondary or tertiary protein structure breaks down, at the temperature above 40 degrees C. In the effects of pH, it is expected that the changes in pH would have an effect on the action of enzymes. Lemon juice helps keep the apple from growing, because its full of ascorbic acid (Vitamin C) and it has a low acidic pH level. But extreme high levels of low pH values can result in a complete loss of enzymes activity thus leaving the apple to brown. The effects of substrate concentration, an enzyme substrate complex is formed when a substrate fits into active of an enzyme. The velocity, the rate of speed, at which the enzymes works will increase until it reaches a maximum. If the substrate is present...
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...Your Full Name: UMUC Biology 102/103 Lab 4: Enzymes INSTRUCTIONS: * On your own and without assistance, complete this Lab 4 Answer Sheet electronically and submit it via the Assignments Folder by the date listed in the Course Schedule (under Syllabus). * To conduct your laboratory exercises, use the Laboratory Manual located under Course Content. Read the introduction and the directions for each exercise/experiment carefully before completing the exercises/experiments and answering the questions. * Save your Lab 4 Answer Sheet in the following format: LastName_Lab4 (e.g., Smith_Lab4). * You should submit your document as a Word (.doc or .docx) or Rich Text Format (.rtf) file for best compatibility. Pre-Lab Questions 1. How could you test to see if an enzyme was completely saturated during an experiment? - Add more substrate and record the rate. If the rate of the reaction is constant, all the enzymes are saturated. 2. List three conditions that would alter the activity of an enzyme. Be specific with your explanation. * Temperature – Cold temperature will cause the enzyme to work slow, hot temperature will cause the enzyme to increase the movement making it less stable. * PH – Difference in range in the PH scale can alter the shape of the enzyme’s active site * Concentration Of Substrate – Less or more of enzymes to substrates ratio will affect the rate of collisions between the two affecting the number of reactions. ...
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...experiment did not support the created hypothesis that the poisonous enzyme PTU (Phenylthiourea) was a competitive inhibitor. In the experiment to test whether PTU was competitive a catechol oxidase substrate was added to the solution to determine it’s inhibition. As more of the substrate was added, no reaction occurred and the substance remained consistently clear. However, the results indicated that the catechol that was used continued to be inhibited by the PTU. Due to this inhibition, catechol oxidase was present in potatoes and the results suggested opposite from the hypothesis. Enzyme inhibitors are compounds that counteract substrate-enzyme interactions. Two classes of enzyme inhibitors are competitive and noncompetitive. Enzyme inhibitors work as one of the major controls in the biological systems. Competitive inhibitors act in the spot that are meant to be for it’s intended substrate. At any given moment enzymes may bind to its inhibitor, the substrate or neither. They are unable to bind to both at the same time. This inhibition competes with the substrate for binding sites on the enzymes. However, a competitive inhibitor is reversible if the concentration of the substrate is raised to high levels while the inhibitor is constant. Noncompetitive inhibitors do not compete for its position in the active site. It decreases the activity of the enzyme and binds equally even if it’s bound to the substrate or not. This causes the conformations of the protein to change....
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