Experiment on the Time Course of an Enzyme Catalysed Reaction

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8th Feb 2020 Chemistry Reference this

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Introduction

Enzymes are proteins which are continuous long chains of amino acids, that are structured in a folded formation that creates the active site. This is completed by a substrate binding to the enzyme to form an enzyme substrate complex (Leech and Newsholme,2013). Once the substrate has fused to the enzyme it produces what are known as reaction products that are created only after the main reaction has taken place and allows the enzyme to be used again for another reaction (Starr, Evers and Starr, 2009). The aim of this experiment is to look at how acid phosphatase can react when placed into different PH concentrations and placed within (pNPP) paranitrophenol phosphate. To see how effective this is at producing a catalytic reaction within a time period.

Abstract

Enzymes are biological catalysts. They can accelerate a wide variety of biochemical responses that can be within a living body or can be seen in a practical experiment (Blackburn, 1976).  Although enzymes are made of proteins, they can also be made up by what is known as a non-protein component. This non-protein part of the enzyme is called a co-factor. In general, the protein component causes an enzyme catalytic reaction (Petsko and Ringe, 2009). Cofactors have the ability to react with metal ions that connect both the enzyme and the substrate together combining with protein to become a catalyst. Cofactors also react with coenzymes when simple proteins that enzymes are made from are unable to do this (Sackheim, 2008) This is because coenzymes are non-proteins coming under the category of organic complexes as cofactors react with organic groups by bonding permanently to the enzyme. (Williams, 2000)

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Enzymes can increase chemical responses by providing an additional reaction direction and can reduce energy when required to do so. The three-dimensional structure of enzymes and bound protein regulate the functioning (Komoda and Matsunaga, n.d.). This then causes a response to occur when the substrate molecule passes into the specific corresponding site which may be an irrelevant part of the protein. The rest of the enzyme acts from time to time as a platform in the active site creating an innovative enzyme-linked substrate. (Illanes, 2008) Therefore, the site gets its properties after the amino acids form it, of which the weak sequential type and the element are fixed to the molecule of the substrate. This suggests that the site is functional only on a substrate. The protein also helps to change the state of the reaction by forming the conditions such as pH, water load and concentration between the site which is completely different from the external configuration (Pelley, Goljan and Pelley, 2011). In general, in reactions, the substrate decreases in more or 2 neighboring substrates to form a larger molecule. Numerous enzymes also have coenzyme in the site which are non-protein molecules, occasionally derived from vitamins, which together help to bind the substrate molecule.

There is a theory that clarify the interaction of this protein substrate: the lock and key model (Bettelheim, Brown and Campbell, 2009). The lock and key model states that enzymes are more rigid, the site precisely adapts to the molecule of the substrate. This can sometimes be explained by a key adjustment and correspond specifically to a lock (Copeland, 2004). On the opposite side, the match pattern obtained indicates that the protein is more versatile, so the site will be slightly deformed. This suggests that the site does not correspond to the substrate molecule, however, as the substrate molecule begins to bind, the site begins to form around to present a closer position.

This closer adjustment distorts the substrate molecule within the site by breaking and reforming the additional bonds, by lengthening them, then producing more to break them so it is possibly added to create the properties (Kuriyan, Konforti and Wemmer, 2012). The site and this closer set contribute to creating a larger product by positioning the 2 molecules in the correct orientation to form a connection between them, pushing them effectively, forming the largest product (Rodwell et al., 2018).

Method

Temperature can a have a huge affect on enzyme activity. This is because, molecules begin to speed up, due to the increase in speed the molecules begin to collide with one another as well as the substrate and the enzyme (A.C. BOWDEN., 2018).  As this is taking place it causes more kinetic energy to be stimulated which then further increases the collusion frequency causing the reaction rate to increase (Gray, 1971). All enzymes have an optimum temperature so they can perform their normal functions (House, 2007). When approaching the optimum temperature this is when catalytic activity is at its prime. A good example of this is humans where the optimum temperature before causing the enzyme to denature is 37°c (Clugston and Flemming, 2013). Beyond this temperature will cause the enzyme activity to gradually decrease, which is what is known as the enzyme becoming denatured  (Scopes, 1993) This happens because there has been a change to the active site of the enzymes structure. Since the active site has changed shape it is no longer able to fit to the substrate resulting in denaturation to occur because, the protein begins to unfold making the enzyme lose its once tertiary construction becoming incapable to catalyse biochemical reactions (Kessel and Ben-Tal, 2018). The enzyme within humans will denature at 40°c.

PH can affect enzyme activity this is because every single enzyme has its own value range of PH that can range between 0- 14 (Griffin, 2019).  A PH of 7 is neutral which the enzyme Trypsin operates at. A PH less than 7 comes under the category of acidic which some enzymes operate at for instance, the enzyme Pepsin is found within the stomach and operates at a low PH of 2 which is extremely strong acid that is why the enzyme Pepsin is found within the stomach as it is a part of the stomach acid to aid in the breakdown of food and digestion and makes the enzyme function at a great velocity. A PH that is higher than 7 is known as a basic or alkaline (Sadava, 2008)

If the incorrect PH is used on an enzyme this will cause a change to take place, editing the physical form of the enzyme mainly changing the active site that effects the enzyme causing denaturation to take place as the enzyme is no longer compatible to the active site (Clugston and Flemming, 2013).   All enzymes are different, they each have their own optimum PH. The reason behind why an enzyme has its own suitable PH is whatever reaction the enzyme is a part of, has its own required PH. Any alterations to PH will cause the intermolecular and intramolecular bonds to simply break apart from one another permanently disfiguring the enzymes shape (Purich, 2010).

Enzyme and substrate concentration can affect enzyme activity because in order for an enzyme- substrate reaction to occur the concentration of enzymes and the substrate should be in equal distribution, otherwise if there is more enzyme being produced this will cause the reaction rate to speed up too which means if the number of enzymes is greater than the substrate the enzymes have nothing to collide with causing the reaction rate to decrease equally this is the same for substrate the more substrate there are than enzymes the left over substrate simply denature due to not being filled by enzymes.

Enzyme catalyzed reactions are liner (Prasad, 2011). However, over time this may change since the reaction rate has reached its optimal level and can no longer react becoming denatured. From this a total amount of product can then be recorded using a graph that then can be plotted against time producing what is known as a curvilinear response, this can be demonstrated on the graph below.

(Figure 1)

From looking at figure 1. I can see the higher the concentration of PNP the higher the absorbance. This is because, enzymes combine to its substrate starting off a catalyzes reaction that then produces products. This time process often takes a fair amount of time depending on the enzyme, and how it releases its products, as the more enzymes available increases how much product is produced as the enzymes combine with the substrate. However, this then depends on the concentration of pNPP paranitrophenol when placed within acid phosphatase becomes hydrolysed forming pNP phosphate (Frey and Northrop, 1999).

figure 2

figure 3

(Figure 4)

From looking at my results figure 2, figure 3 and figure 4. It can be clearly seen, the more alkaline the PH the quicker the reaction took place. This is because, proteins have net charges, either ionic or covalent. Which can either attract or repel one another. This then has an effect on the tertiary/ quaternary structure that most proteins are made from (Talwar, Hasnain and Sarin, 2016). A protein that has a surrounding PH and due to the acid or base being to strong or weak can destroy the PH of the protein causing denaturation to take place.

Conclusion

From looking at my results from each experiment it can be concluded the higher the concentration of pNPP (paranitrophenol phosphate) caused a delay on how long a reaction would take place when mixed with phosphate. Furthermore, from looking at figures 2,3,4 shows the higher the PH the less time it took for a catalyzed reactions to take place.

References

Introduction

Enzymes are proteins which are continuous long chains of amino acids, that are structured in a folded formation that creates the active site. This is completed by a substrate binding to the enzyme to form an enzyme substrate complex (Leech and Newsholme,2013). Once the substrate has fused to the enzyme it produces what are known as reaction products that are created only after the main reaction has taken place and allows the enzyme to be used again for another reaction (Starr, Evers and Starr, 2009). The aim of this experiment is to look at how acid phosphatase can react when placed into different PH concentrations and placed within (pNPP) paranitrophenol phosphate. To see how effective this is at producing a catalytic reaction within a time period.

Abstract

Enzymes are biological catalysts. They can accelerate a wide variety of biochemical responses that can be within a living body or can be seen in a practical experiment (Blackburn, 1976).  Although enzymes are made of proteins, they can also be made up by what is known as a non-protein component. This non-protein part of the enzyme is called a co-factor. In general, the protein component causes an enzyme catalytic reaction (Petsko and Ringe, 2009). Cofactors have the ability to react with metal ions that connect both the enzyme and the substrate together combining with protein to become a catalyst. Cofactors also react with coenzymes when simple proteins that enzymes are made from are unable to do this (Sackheim, 2008) This is because coenzymes are non-proteins coming under the category of organic complexes as cofactors react with organic groups by bonding permanently to the enzyme. (Williams, 2000)

Enzymes can increase chemical responses by providing an additional reaction direction and can reduce energy when required to do so. The three-dimensional structure of enzymes and bound protein regulate the functioning (Komoda and Matsunaga, n.d.). This then causes a response to occur when the substrate molecule passes into the specific corresponding site which may be an irrelevant part of the protein. The rest of the enzyme acts from time to time as a platform in the active site creating an innovative enzyme-linked substrate. (Illanes, 2008) Therefore, the site gets its properties after the amino acids form it, of which the weak sequential type and the element are fixed to the molecule of the substrate. This suggests that the site is functional only on a substrate. The protein also helps to change the state of the reaction by forming the conditions such as pH, water load and concentration between the site which is completely different from the external configuration (Pelley, Goljan and Pelley, 2011). In general, in reactions, the substrate decreases in more or 2 neighboring substrates to form a larger molecule. Numerous enzymes also have coenzyme in the site which are non-protein molecules, occasionally derived from vitamins, which together help to bind the substrate molecule.

There is a theory that clarify the interaction of this protein substrate: the lock and key model (Bettelheim, Brown and Campbell, 2009). The lock and key model states that enzymes are more rigid, the site precisely adapts to the molecule of the substrate. This can sometimes be explained by a key adjustment and correspond specifically to a lock (Copeland, 2004). On the opposite side, the match pattern obtained indicates that the protein is more versatile, so the site will be slightly deformed. This suggests that the site does not correspond to the substrate molecule, however, as the substrate molecule begins to bind, the site begins to form around to present a closer position.

This closer adjustment distorts the substrate molecule within the site by breaking and reforming the additional bonds, by lengthening them, then producing more to break them so it is possibly added to create the properties (Kuriyan, Konforti and Wemmer, 2012). The site and this closer set contribute to creating a larger product by positioning the 2 molecules in the correct orientation to form a connection between them, pushing them effectively, forming the largest product (Rodwell et al., 2018).

Method

Temperature can a have a huge affect on enzyme activity. This is because, molecules begin to speed up, due to the increase in speed the molecules begin to collide with one another as well as the substrate and the enzyme (A.C. BOWDEN., 2018).  As this is taking place it causes more kinetic energy to be stimulated which then further increases the collusion frequency causing the reaction rate to increase (Gray, 1971). All enzymes have an optimum temperature so they can perform their normal functions (House, 2007). When approaching the optimum temperature this is when catalytic activity is at its prime. A good example of this is humans where the optimum temperature before causing the enzyme to denature is 37°c (Clugston and Flemming, 2013). Beyond this temperature will cause the enzyme activity to gradually decrease, which is what is known as the enzyme becoming denatured  (Scopes, 1993) This happens because there has been a change to the active site of the enzymes structure. Since the active site has changed shape it is no longer able to fit to the substrate resulting in denaturation to occur because, the protein begins to unfold making the enzyme lose its once tertiary construction becoming incapable to catalyse biochemical reactions (Kessel and Ben-Tal, 2018). The enzyme within humans will denature at 40°c.

PH can affect enzyme activity this is because every single enzyme has its own value range of PH that can range between 0- 14 (Griffin, 2019).  A PH of 7 is neutral which the enzyme Trypsin operates at. A PH less than 7 comes under the category of acidic which some enzymes operate at for instance, the enzyme Pepsin is found within the stomach and operates at a low PH of 2 which is extremely strong acid that is why the enzyme Pepsin is found within the stomach as it is a part of the stomach acid to aid in the breakdown of food and digestion and makes the enzyme function at a great velocity. A PH that is higher than 7 is known as a basic or alkaline (Sadava, 2008)

If the incorrect PH is used on an enzyme this will cause a change to take place, editing the physical form of the enzyme mainly changing the active site that effects the enzyme causing denaturation to take place as the enzyme is no longer compatible to the active site (Clugston and Flemming, 2013).   All enzymes are different, they each have their own optimum PH. The reason behind why an enzyme has its own suitable PH is whatever reaction the enzyme is a part of, has its own required PH. Any alterations to PH will cause the intermolecular and intramolecular bonds to simply break apart from one another permanently disfiguring the enzymes shape (Purich, 2010).

Enzyme and substrate concentration can affect enzyme activity because in order for an enzyme- substrate reaction to occur the concentration of enzymes and the substrate should be in equal distribution, otherwise if there is more enzyme being produced this will cause the reaction rate to speed up too which means if the number of enzymes is greater than the substrate the enzymes have nothing to collide with causing the reaction rate to decrease equally this is the same for substrate the more substrate there are than enzymes the left over substrate simply denature due to not being filled by enzymes.

Enzyme catalyzed reactions are liner (Prasad, 2011). However, over time this may change since the reaction rate has reached its optimal level and can no longer react becoming denatured. From this a total amount of product can then be recorded using a graph that then can be plotted against time producing what is known as a curvilinear response, this can be demonstrated on the graph below.

(Figure 1)

From looking at figure 1. I can see the higher the concentration of PNP the higher the absorbance. This is because, enzymes combine to its substrate starting off a catalyzes reaction that then produces products. This time process often takes a fair amount of time depending on the enzyme, and how it releases its products, as the more enzymes available increases how much product is produced as the enzymes combine with the substrate. However, this then depends on the concentration of pNPP paranitrophenol when placed within acid phosphatase becomes hydrolysed forming pNP phosphate (Frey and Northrop, 1999).

figure 2

figure 3

(Figure 4)

From looking at my results figure 2, figure 3 and figure 4. It can be clearly seen, the more alkaline the PH the quicker the reaction took place. This is because, proteins have net charges, either ionic or covalent. Which can either attract or repel one another. This then has an effect on the tertiary/ quaternary structure that most proteins are made from (Talwar, Hasnain and Sarin, 2016). A protein that has a surrounding PH and due to the acid or base being to strong or weak can destroy the PH of the protein causing denaturation to take place.

Conclusion

From looking at my results from each experiment it can be concluded the higher the concentration of pNPP (paranitrophenol phosphate) caused a delay on how long a reaction would take place when mixed with phosphate. Furthermore, from looking at figures 2,3,4 shows the higher the PH the less time it took for a catalyzed reactions to take place.

References

  • A.C. BOWDEN. (2018). FUNDAMENTALS OF ENZYME KINETICS. [S.l.]: ED-TECH.
  • Bettelheim, F., Brown, W. and Campbell, M. (2009). General, organic & biochemistry. Australia: Brooks/Cole Cengage Learning.
  • Blackburn, S. (1976). Enzyme structure and function. New York: Dekker.
  • Clugston, M. and Flemming, R. (2013). Advanced chemistry. Oxford: Oxford University Press.
  • Copeland, R. (2004). Enzymes. New York, NY: John Wiley & Sons.
  • Gray, C. (1971). Enzyme-catalysed reactions. London: Van Nostrand Reinhold.
  • House, J. (2007). Principles of chemical kinetics. Oxford: Academic.
  • Illanes, A. (2008). Enzyme biocatalysis. [Dordrecht]: Springer.
  • Frey, P. and Northrop, D. (1999). Enzymatic mechanisms. Amsterdam: IOS Press.
  • Griffin, M. (2019). The pH scale. Gareth Stevens.
  • Kessel, A. and Ben-Tal, N. (2018). Introduction to proteins. Taylor and Francis group.
  • Komoda, T. and Matsunaga, T. (n.d.). Biochemistry for Medical Professionals.
  • Kuriyan, J., Konforti, B. and Wemmer, D. (2012). The molecules of life. New York: Garland Science.
  • Leech, A. and Newsholme, E. (2013). Functional biochemistry in health and disease. Hoboken, N.J.: Wiley.
  • Pelley, J., Goljan, E. and Pelley, J. (2011). Biochemistry. Philadelphia, PA: Mosby/Elsevier.
  • Purich, D. (2010). Enzyme kinetics. Amsterdam: Elsevier/Academic Press.
  • Petsko, G. and Ringe, D. (2009). Protein structure and function. Oxford [u.a]: Oxford Univ. Press.
  • Prasad, N. (2011). Enzyme technology. New Delhi: PHI Learning.
  • Rodwell, V., Harper, H., Bender, D., Botham, K., Kennelly, P., Weil, P., Smoleński, R. and Gross, P. (2018). Biochemia Harpera ilustrowana. Warszawa: PZWL Wydawnictwo Lekarskie.
  • Sackheim, G. (2008). An introduction to chemistry for biology students. San Francisco, Calif.: Benjamin Cummings.
  • Sadava, D. (2008). Life, the science of biology. Sunderland, MA: Sinauer Associates.
  • Scopes, R. (1993). Protein purification. New York: Springer-Vlg.
  • Starr, C., Evers, C. and Starr, L. (2009). Biology. 5th ed. Van Nostrand Reinhold.
  • alwar, G., Hasnain, S. and Sarin, S. (2016). Textbook of biochemistry, biotechnology, allied and molecular medicine. [Place of publication not identified]: Prentice-Hall of India.
  • Williams, G. (2000). Advanced biology for you. Cheltenham: Thornes.

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