Title: Preparation and Assay of Phenolase and Peroxidase from Sweet and Irish Potato.

Aim: To determine the degree of specificity of the enzymes Polyphenol Oxidase and Peroxidase; to determine the effect of inhibitors on enzyme activity and to examine the effect of enzyme activity on the rate of breakdown of the substrates provided.

Theory: Enzymes are catalysts. A catalyst is a substance which speeds up the rate of a chemical reaction without itself being used up. Most enzymes are proteins. The catalytic activity of enzymes is based on the precision of the protein conformation of the enzyme. In particular, this specificity depends upon the shape of a small part of the enzyme molecule where the enzyme actually comes into contact with the substrate molecule, called the active site. This is a small cleft in the surface of the enzyme where certain amino acid residues are exposed. These residues contain substituent groups that bind to the substrate and catalyse its chemical transformation. The substrate molecule fits into the active site and interacts with these amino acids by ionic and hydrogen bonding, forming an enzyme-substrate complex. A reaction then occurs, and the product leaves the active site. If an enzyme is broken down into its component amino acids, its catalytic activity is always destroyed. Thus the primary, secondary, tertiary, and quaternary structures of enzymes proteins are essential to their catalytic activity. One of the aims of this experiment is to examine the effect of enzyme activity on the rate of breakdown of the substrates provided. This will in turn be used to determine how specific enzymes are their substrates since the substrates which react most rapidly with the enzyme will be the most complementary. Enzymes are highly specific, readily discriminating between substrates with quite similar structures. This is due to the chemical reactions that take place between substrates and enzymes functional groups within the active site of the enzyme.

Procedure: This lab was divided into two parts, the first of which utilized the enzyme Polyphenol Oxidase (Phenolase) and the second of which utilized the enzyme, Peroxidase. In the first part of the lab, the effect of enzymes on the rate of substrate breakdown was examined. Also, the effect of inhibitors on enzyme activity was examined. For this section of the lab, 5 test tubes were first prepared, each containing 2 mL of the substrate, Catechol. 2 mL of water was added to the first test tube. 2 mL of the enzyme, Phenolase was added to the second test tube alone. 2 mL of inhibitor was added to the third test tube alone. 2 mL of enzyme and 2 mL of inhibitor were added to the fourth test tube and to the fifth test tube

was added 2 mL of boiled enzyme. Observations of any colour changes and the time taken for these colour changes to occur were made and recorded. Still in the first section of the lab, 3 other test tubes were prepared, each containing a different substrate. The first of these 3 test tubes contained 2.0 mL of Caffeic acid, the substrate, and 2.0 mL of the enzyme, Phenolase. The second of these 3 test tubes contained 2.0 mL of the substrate, Pyrogallol and 2.0 mL of the enzyme, Phenolase. The third of these 3 test tubes contained 2.0 mL of the substrate and 2.0 mL of the enzyme, Phenolase. These three test tubes were observed immediately after mixing the substrates with the Phenolase. These observations were recorded. The second part of the lab dealt with the enzyme Peroxidase and its oxidation of a variety of phenols in the presence of hydrogen peroxide. It also dealt with the effects of inhibitors on enzyme activity by adding 2 drops of inhibitor to a particular substrate and then adding the enzyme after. It examined the degree of specificity of the enzyme in use by adding the said enzyme to a number of substrates which were all similar in structure to each other and then observing the effects of the enzyme on the different substrates. For this section of the lab, 5 test tubes were prepared, each containing a different substrate. The first test tube contained 2.0 mL of the substrate, caffeic acid, to which was then added 1.0 mL of the enzyme, peroxidase and 3 drops of hydrogen peroxide. Observations of the effects of the enzyme on the substrate were made and recorded. The second test tube contained 2.0 mL of the substrate, pyrogallol, to which was then added 1.0 mL of enzyme and 3 drops of hydrogen peroxide. Observations of the effects of the enzyme on the substrate were made and recorded. The third test tube contained 2.0 mL of the enzyme, catechol to which was then added 1.0 mL of enzyme and 3 drops of hydrogen peroxide. Observations of the effects of the enzyme on the substrate were made and recorded. The fourth test tube contained 2.0 mL of the substrate, tyrosine, to which was then added 1.0 mL of enzyme and 3 drops of hydrogen peroxide. Observations of the effects of the enzyme on the substrate were made and recorded. The fifth test tube contained 2.0 mL of the substrate, guiaicol, to which was then added 1.0 mL of enzyme, 3 drops of hydrogen peroxide and 2 drops of inhibitor. Observations of the effects of the enzyme on the substrate in the presence of inhibitor were made and recorded.

Results: Table 1: Observations of the effects of the enzyme Phenolase on the substrate Catechol and other substrates.

Reaction Mixture Catechol and Water

Observations Upon adding water to the Catechol, it remained colourless. The solution showed no change in appearance after a long period of time. Upon adding the enzyme Phenolase, to the Catechol the contents of the tube turned a pale orange colour. After some time, the contents turned dark brown. Upon adding inhibitor to the substrate, the contents of the tube remained colourless. The solution showed no change in appearance or colour, after a long, period of time. Upon adding Inhibitor and then enzyme, the solution remained colourless. After a long period of time, no change in appearance of the contents of the tube was observed. A white precipitate was observed in a colourless solution. Upon adding the Phenolase to the Caffeic acid, a pale peach colour was observed which over time slowly turned to a pale orange colour. Upon adding the Phenolase to the Pyrogallol, a pale peach colour was observed which rapidly changed to a deep yellow colour. Upon adding the Phenolase to the Tyrosine, a pale peach colour was observed. After a long period of time no change was observed indicating little or no reaction.

Catechol and Phenolase

Catechol and Inhibitor

Catechol, Inhibitor and Enzyme

Catechol and Boiled Enzyme

Caffeic acid and Phenolase

Pyrogallol and Phenolase

Tyrosine and Phenolase

Table 2: Observations of the Effects of Peroxidase on Different Substrates Reaction Mixture Observations

Caffeic acid, Peroxidase and Hydrogen Peroxide

Upon adding the Peroxidase to the Caffeic acid and Peroxide mixture, the contents of the tube were initially colourless. As time progressed the mixture went from colourless to pale orange and a thick layer of fine bubbles were observed at the surface of the mixture. Upon adding the Peroxidase to the Pyrogallol and Peroxide mixture, the contents of the tube were initially colourless. The mixture then quickly went from colourless to a yellow orange brown colour. A thick layer of fine bubbles were observed at the surface of the mixture. Upon adding the Peroxidase to the Catechol and Peroxide mixture, the contents of the tube were initially colourless. The mixture then went rapidly from colourless to pale orange to a darker, burnt orange to a red brown colour. A layer of fine bubbles were observed at the surface of the mixture. Upon adding the Peroxidase to the Tyrosine and Peroxide mixture, the contents of the tube were initially colourless. The mixture then went slowly form colourless to peach. A layer of fine bubbles were observed at the surface of the mixture. Upon adding the Peroxidase to the Guiaicol, Peroxide and Inhibitor mixture the contents of the tube were initially colourless. The mixture then went rapidly from colourless to red to a dark, red brown colour. A layer of fine bubbles were seen at the surface of the mixture.

Pyrogallol, Peroxidase and Hydrogen Peroxide

Catechol, Peroxidase and Hydrogen Peroxide

Tyrosine, Peroxidase and Hydrogen Peroxide

Guiaicol, Peroxidase and Hydrogen Peroxide, Inhibitor

Discussion: The purpose of this lab was to examine the function of enzymes in their conversion of substrates to their products, to examine the effect of an inhibitor on an enzyme and to determine the degree of specificity of certain types of enzymes with respect their substrates,

In the first part of the lab, the enzyme under study was the enzyme, Polyphenol Oxidase (Phenolase). The substrates of Polyphenoloxidases are mono-, di- , tri and polyhydroxyphenols which are oxidised to their corresponding quinones by the enzyme. Polyphenoloxidases catalyse the o-hydroxylation of monophenols to o-diphenols. They can also further catalyse the oxidation of o-diphenols to produce o-quinones. It is the rapid polymerization of o-quinones to produce black, brown or red pigments (polyphenols) that is the cause of food browning. The first part of the lab dealt with the effect of enzyme activity on the dihydroxyphenol substrate, Catechol. The structural formula of Catechol is:

A mixture of Catechol and water yielded no visible reaction. Upon addition of the Phenolase enzyme however, a change was observed as the mixture changed from colourless, to pale orange to dark brown. This was due to the Phenolase enzyme oxidising the Catechol to its benzoquinone derivative which is a reddish brown melanoid pigment. In the third test tube, inhibitor was added to the substrate alone without any enzyme and no reaction was observed. The purpose of preparing this tube was to show that the inhibitor does not have any direct effect on the substrate and does not oxidise it. The fourth test tube had a substrate and inhibitor mixture to which was then added the enzyme Phenolase. No change was observed in this mixture. Since the test tube which contained the enzyme and substrate had undergone a colour change thus indicating that a reaction had taken place and since the test tube which contained the inhibitor and substrate alone had undergone no visible colour change, it can be concluded that the lack of reaction in the fourth test tube was due to the inhibitor. No other reactants were present to prevent the oxidisation of the substrate besides the inhibitor. The enzyme was responsible for the oxidation of the substrate in the second test tube. The fifth test tube had a reaction mixture comprised of the substrate Catechol and denatured enzymes which had been prepared by boiling. No change in the substrate was observed i.e a dark brown colour had not appeared after some time indicating that no reaction had taken place. The purpose of this tube was to show how important the structure of an enzyme is to its function. The substrate specificity of an enzyme is determined by the properties and spatial arrangement of the amino acid residues forming the active site. Boiling enzymes can disrupt the hydrogen, van der Waals bonds, hydrophobic interactions and electrostatic interactions that maintain the tertiary and quaternary structure of enzymes. Boiling will thus affect the spatial arrangement of the

exposed residues in the acitive site of the enzyme which is needed in order for the enzyme to bind to the substrate. Since enzymes are highly specific, slight changes in the active site will determine whether or not the substrate can bind to the active site or whether or not the enzyme will be able to carry out its function. Three other test tubes were prepared in the first part of the lab, each containing a different substrate but the same Phenolase enzyme. The first of these three tubes contained Caffeic acid. A pale peach colour was observed which over time changed to a pale orange colour. The second contained Pyrogallol which rapidly changed from a pale peach colour to a deep yellow colour. The third of these contained Tyrosine which underwent no reaction with the enzyme Phenolase. From these results it can be seen that Pyrogallol was oxidised at the fastest rate and underwent the most drastic colour change. The structure of Pyrogallol is shown below:

The position of the hydroxyl substituents are integral in the formation of the enzyme-substrate complex and the fact that there are two possible 2- positions on the aromatic ring to which hydroxyl groups are bonded increases the chances of this happening. The structure of Pyrogallol is also quite similar to the structure of Catechol. Caffeic acid did not react as quickly as the Pyrogallol and only reacted to a small extent, the colour change not being very drastic. The structure of Caffeic acid is shown below:

There is a hydroxyl group at the 2- and 3- positions of the aromatic ring and a bulky side group at the 5- position. Relative to Catechol the positions of the hydroxyl groups are significantly different. Enzymes are very substrate specific so would thus not react to a large extent with a substrate that was so different from a substrate which was so readily oxidised by the Phenolase.

The bulky side group at the 5- position would also prevent proper binding to the active site. It would serve as a steric hindrance to the enzyme. Tyrosine did not react at all with the enzyme. Its structure is shown below:

Whilst there is a hydroxyl group at the 1- position which is perhaps able to bind to the active site of the enzyme, the side group at the 4-position of the aromatic ring would act as a steric hindrance to binding to the active site of the substrate. This explains the lack of reaction of Tyrosine with the enzyme, Phenolase. The results produced by the last three tubes indicate that Phenolase shows relative specificity. The more similar in structure the substrates were to Catechol, the more rapidly they reacted with the enzyme. The second part of the experiment dealt with the enzyme Peroxidase. The enzyme peroxidase catalyses the oxidation of a variety of phenols and ceratin aromatic amines in the presence of hydrogen peroxide. For this part of the lab five test tubes were prepared, four of which contained a different substrate, hydrogen peroxide and the enzyme, Peroxidase. The fifth contained a different substrate, hydrogen peroxide, enzyme and inhibitor. The first test tube contained Caffeic acid which did not react rapidly with the enzyme and which did not undergo a drastic colour change. It did however undergo some reaction since it went from colourless to pale orange. The bubbles that were seen at the surface of the mixture were bubbles of oxygen. The second test tube contained Pyrogallol which reacted rapidly with the enzyme Peroxidase to form a dark, yellow brown product. The active site of the enzyme Peroxidase was perhaps more specific to the structure of Pyrogallol than it was to that of Caffeic acid. The bubbles were again oxygen. The third test tube contained Catechol which reacted rapidly with the enzyme Peroxidase to form a dark, red brown product. Catechol was thus more complementary to the active site of the enzyme than Pyrogallol or Caffeic acid. The bubbles at the surface were again oxygen. The fourth test tube contained Tyrosine which only reacted to a small or negligible extent with the enzyme, Peroxidase. The colour change was from colourless to peach and was very difficult

to observe. The bulky substituent group at the 4-position of the aromatic ring prevented the enzyme from oxidising the substrate in this case as well. The bubbles observed at the surface were oxgen. These observations show that Catechol was the substrate most complementary to the active site of the enzyme Peroxidase with Pyrogallol being the second most complementary. In the fifth test tube, the substrate that was used was Guiacol. Hydrogen peroxide, the enzyme Peroxidase and inhibitor were used with this substrate. It was expected that no reaction would take place since inhibitor had been included in the reaction mixture. However, Guiacol reacted rapidly with the substrate and a dark, red brown product was produced. The amount of inhibitor added (2 drops) was not adequate for its effects to be seen. This part of the lab with respect to the tube containing Guiacol and inhibitor could have therefore been improve by adding more inhibitor than merely 2 drops. The substrate Pyrogallol decolourises in the presence of light. This is a possible source of error since results were largely based on colour changes of the reaction mixtures and it was required that colour changes be entirely due to the enzymes that were used. When using the inhibitor, a precaution was taken to add the inhibitor before the enzyme to the substrate. As was shown in the first part of the lab, the inhibitor does not have a direct effect on the substrate so would not interfere with the enzyme acting on the substrate in this respect. It only directly affects the enzyme. If the enzyme was added to the substrate before the inhibitor some substrate breakdown would take place leading to confusion of results and wrong conclusions.

Additional Discusion: 1)To what class of enzymes does a) phenolase and b) peroxidase belong? Both of these enzymes are oxidoreductases since they both react with the same substrates in a similar manner and both involve the transfer of electrons. 2)Explain what happens on boiling the enzyme preparation. Boiling temperature is much higher than the optimum temperature of enzymes. High temperatures change the shape of the enzyme molecule. Enzymes are globular proteins. Their tertiary structure is maintained by bonds between the amino acid R-groups of a polypeptide chain. High temperatures cause these bonds to break, damaging the tertiary structure of the protein. If the three dimensional shape of the active site has been altered, substrate molecules will be unable to combine with it. The irreversible change in the structure of the enzyme at high temperatures is called denaturation.

3)Give examplesof phenolase activity in everyday situations. How do we try to control such activity? Enzymatic browning of fruits and some vegetables is a chemical process that occurs in everyday situations. It involves phenolase breakdown of polyhydroxyphenols such as Catechol to their brown coloured quinones. Enzymatic browning can be prevented by sprinkling lemon juice on the freshly cut fruits and vegetables. The ascorbic acid in the lemon juice acts as an inhibitor preventing the breakdown of the substrate to the brown coloured quinone product. 4)What class of inhibitor was used in your experiment? Competitive inhibitors were used since with competitive inhibitors the degree of inhibition depends on the relative concentration of substrate and inhibitor. Increasing the concentration of the substrate will reduce the effect of the competitive inhibitor which is what happened in this lab. The much greater concentration of substrate made the effects of the inhibitor negligible.

References:
David Hames, Nigel Hooper. Instant Notes in Biochemistry. New York: Taylor and Francis Group, 2005. David L. Nelson, Michael M. Cox. Lehninger Principles of Biochemistry. New York: W. H. Freeman and Company, 2008. T. W. Graham Solomons, Craig B. Fryhle. Organic Chemistry. New Jersey: John Wiley & Sons, 2006.