Study of Sterospecificity in Mushroom Tyrosinase

Enzymes are types of proteins that work as a substance to help speed up a chemical reaction (Madar & Windelspecht, 104). There are three factors that help enzyme activity increase in speed. The three factors that speed up the activity of enzymes are concentration, an increase in temperature, and a preferred pH environment. Whether or not the reaction continues to move forward is not up to the enzyme, instead the reaction is dependent on a reaction’s free energy. These enzymatic reactions have reactants referred to as substrates. Enzymes do much more than create substrates; enzymes actually work with the substrate in a reaction (Madar &Windelspecht, 106). For reactions in a cell it is

The role of an enzyme is to catalyse reactions within a cell. The enzyme present in a potato (Solanum Tuberosum) is catechol oxidase. In this experiment, the enzyme activity was tested under different temperature and pH conditions. The objective of this experiment was to determine the ideal conditions under which catechol oxidase catalyses reactions. In order to do this, catechol was catalyzed by catechol oxidase into benzoquinone at diverse temperatures and pH values. The enzyme was exposed to its new environment for 5 minutes before the absorbance of the catechol oxidase was measured at 420 nm using a spectrophotometer. The use of a spectrophotometer was crucial for the collection of data in this experiment. When exposed to hot and cold temperatures, some enzymes were found to denature causing the activity to decrease. Similarly, when the pH was too high or low, then the catechol oxidase enzyme experienced a significant decrease in activity. It can be concluded after completing this experiment that the optimal pH for catechol oxidase is 7 and that the prime temperature is 20º C. Due to the fact that the catechol oxidase was only tested under several different temperatures and pH values, it is always possible to get a more precise result by decreasing the increments between the test values. However, our experiment was able to produce accurate results as to the

The purpose of the study was to detect the rates of hyphal tip growth and nuclei position of N. crassa in influence of drug medium, Taxol. The results of the experiments show that the rate of hyphal tip growth and nuclei position are much lower in drug medium than in the control. Taxol have different effect on growth of tip and distance travelled by nuclei. Moreover, the rate at which the nuclei displace and growth of the tips are dependent on the medium and not on each other. Thus, the results conclude that drug effected fungus affects the tip growth rate and nuclei displacement.

Tyrosinase is an enzyme found in Portabella mushrooms. Tyrosinase is the enzyme most responsible for the brown color of Portabella mushrooms, as it is found in larger amounts than other pigmentations. Tyrosinase is also found in Tyrosinase vesicles in the cell wall of Portabella mushrooms. Tyrosinase plays a huge role in Portabella mushrooms in that it is active in most chemical reactions including oxidation, phosphorylation and other important mechanisms within the cell. The journal article examines the biochemical characteristics of Tyrosinase in Portabella mushrooms by specifically extracting it from the gill tissue. In order to effectively examine Tyrosinase, purification of this enzyme was to be extracted which

After the conclusion of our experiment we refuted our original hypothesis of, “If the type of fungus (parasite or saprophyte) affects the enzyme activity, and you measure enzymes in lion’s mane an enoki, then the lions mane will have less enzyme activity? ” because we found the opposite was apparent in our experiment. While we though the Lion's mane mushroom (parasite) would have less enzyme activity than that of the Enoki mushroom (saprophyte) because the Lion's mane mushroom grows on a living hardwood tree, while the Enoki grows on a dead hardwood tree. That we saw was that The Lion's mane (parasite) which grows on a living hardwood tree, had more enzyme activity than the Enoki (saprophyte) which grows on a dead hardwood tree.

Background: Catalysts are substances that increase the rate of a reaction by providing a different mechanism for the reaction to go through in order to lower the activation energy and are unchanged by the end of the reaction. Enzymes, a specific type of catalyst, are proteins that increase the rate of reaction by converting a substrate to a product in biological processes (Burns, 2002). Enzymes are never used up, but are consistently recycled in order to catalyze many reactions in a certain amount of time. Every enzyme has a specific shape due to the sequence of amino acids, which results in the ability of only a specific substrate to bind to it, whilst others are rejected.

When an enzyme pairs with a substrate there are one of two ways this may occur. The lock and key model refers to a reaction in which the substrate will perfectly fit the active site of an enzyme, while the induced fit model involves a conformational change of both the enzyme and the substrate (Ophardt, 2003). The overall reaction is summarized by the following: initiation is characterized by substrate binding the enzyme at its active site and forming the enzyme-substrate complex at a rate of k1 over k-1 (forward and reverse rates respectively), this is followed by formation/dissociation of the enzyme with product at a rate of k2 (Figure 1) (ChemWiki, 2013). This reaction highlights the basis of enzyme kinetics in that an enzyme and a substrate can progress to a dissociated enzyme and a product, or move in reverse and never form a product. There are also more complicated instances in which there exists an enzyme-product complex or an enzyme-intermediate complex, though these will not be discussed further

Our goal in this experiment is to determine the reaction rate and specific activity of the protein extract. Also, the purpose of this lab is to characterize Vmax and Km for catechol and L-Dopa. Along with, determining whether the mode of inhibition by PTU is competitive or non-competitive? We performed a spectrometry analysis in order to determine the reaction rate of PPO, specific activity, and substrate- enzyme relationships among L-Dopa, catechol, and PTU. The reaction rate of polyphenol oxidase determined to be 0.04932957 μmin/min. By incoporating Beer’s Law, the specific activity of our protein extract was 0.2517127 μm/mgmin.In order to determine Vmax and Km for catechol and L-dopa we had to take averages from 3 dfferent groups. The Vmax for L-dopa determined to be 14.27, the ½ Vmax was 7.134, and the Km value was 0.485. The Vmax for catechol determined to be 10.00, ½ Vmax was 5.33, and the Km value was 0.38. The mode of inhibition by PTU determined to be non- competitive. The graph shows a rough hyperbolic trend with the slop increasing and then slowly making a curve towards the end for the catechol substrate concentration. However, the reaction with catechol and PTU portrayes a flat line. This is due to the fact that the increasing amount of substrate concentration had no effect on the reaction at all. Therefore, PTU was inhibiting the

3b) A non-linear response is observed here because the binding of enzyme to substrate does not follow simple pseudo-first-order kinetics, but instead there is a two-step process involving rate-limiting changes in protein conformation. These findings support the previously proposed model of substrate binding, in that there is a fast step of enzyme binding to substrate, followed by a slow conformational change in the enzyme once bound to substrate. The plateau in the observed rates indicates the maximal rate at which enzyme can bind to substrate, with Abl binding with a maximal rate of 20 s-1 and Src binding with a maximal rate of 2 s-1.

Enzymes are biological catalysts that speed up the rate of a chemical reaction, without being absorbed. Enzymes temporarily bind to substrates in order to lower the activation energy. Activation energy is threshold that must me crossed in order for a reaction to occur before successful collisions give rise to products (Hardin, 2012). Several factors influence the rate at which enzymatic activities occur, such as the concentration of substrate molecules, temperature, inhibitors, and pH. Enzyme kinetics is the study of the rate at which enzymes work, which is a function of the

Tyrosinase and DOPA are key elements in making melanin. In order to model this reaction, we obtained the enzyme from a 15 g peeled potato and placed the chilled enzyme into five pairs of tests tubes with different pHs to measure the amount of melanin produced. The blank test tubes consisted of 4.9 ml of pH 6.0 phosphate buffer in the blanks, 0.1 ml tyrosinase extract, and a pH of 3,5,7,9 or 11. Each reacting test tube consisted of 3.9 ml of pH 6.0 phosphate buffer, 0.1 ml tyrosinase extract, pH of 3,5,7,9 or 11 and 1.0 ml of DOPA. Each test tube was measured with a spectrophotometer at a wavelength at 450 nm at time 0 and every two minutes for fourteen minutes. A blank was put in before measuring each test tube with the DOPA. After

Researchers endeavored to isolate and purify isoforms of tyrosinase within a molecular weight varying from 50 to 230 kDa. The isolation of an isoform exhibiting a molecular weight of 70 kDa was achieved. However the ability to recovery other isoforms was not as successful due to the large quantities of active latent enzymes present and the employment of denaturing SDS reagent. In addition, the assay operated with SDS illustrated latent activity above pH 5.5, whereas below pH 5.5 no latent activity was observed. Overall the chromatography technique employed was effective in the isolation of isoforms from tyrosinase. However due to latent activity,

As the substrate binds to the enzyme, it causes a conformational change in the active site, which stresses the substrate, weakening the bonds and thus, reducing the activation energy required for the reaction to occur. When the substrates form into products, they are still bound to the active site and when the reaction has completed, the products are released, leaving the active site available for substrates to bind again. The enzyme then reverts back to its original shape. (BBC Science)

Mushrooms were known and consumed as food in ceremonies as far back as prehistoric times. Throughout the history, mushrooms were used in different cultures for their medicinal and tonic properties (Gunde-Cimerman, 1999). The longest tradition of using edible mushrooms for medicinal purposes is in China. Mushrooms represent a major and as yet largely untapped source of powerful new pharmaceutical products. The most significant medicinal effect of mushrooms and their metabolites that attracted the attention of the public in the recent years is their antitumour property. Many pharmaceutical substances with potent and unique properties were isolated from mushrooms and distributed worldwide. Many of them are not strictly pharmaceutical products but represent the novel class of dietary supplements of approximately 10,000 known species of mushrooms, 2000 are safe and 300 have significant pharmacological properties (Hobbs, 1995, Miles and Chang, 1997).

Aloesin is a derivative of aloe vera plant.(21) Competitive inhibitor at binding site of tyrosinase and DOPA (3,4-dihydroxyphenylalanine) oxidase is its major mechanism in dose-dependent manner.(21, 24)