Analyzing Pyrophosphate Enzyme Activity

Introduction

Pyrophosphatase (PPase) has been known to be a byproduct of biosynthetic reactions, which is exposed to hydrolysis pyrophosphate. Pyrophosphate is inorganic, and therefore, when it is exposed to reaction through hydrolysis, it forms orthophosphate. It is an enzyme that is used to catalyze the process of conversion of a molecule of pyrophosphate to two phosphate ions. It can also be used in the conversion of hydrolysis of inorganic pyrophosphate to come up with orthophosphate. In short, it is a catalyst that is used in the formation of orthophosphate through the reaction of hydrolysis of inorganic pyrophosphate. The reaction of the pyrophosphatase formula is P2O7-4 + H2O -> 2HPO4-2. The reaction of pyrophosphatase realizes energy, and it cannot be reversed; therefore, it is capable of driving the biochemical reactions to a conclusion.

However, pyrophosphatase is produced in several metabolic reactions, such as the activation of amino acid and the reaction of synthetic products through hydrolyzing of the pyrophosphate to form a product. The phosphate produced through the reaction controls several reactions that occur in biochemical. This is because the energy produced during the reaction is required for the biochemical process or reaction to occur (Pynes & Younathan$, 2012, P. 15). However, pyrophosphatase enzyme kinetics is the pathway that facilitates the reaction of pyrophosphatase through hydrolysis. Kinetic is crystalized inorganic phosphate, which comes from bakers’ yeasts. It is applied in the reaction so that the process can be faster and also to mutant the process.

Materials And Methods

In order to complete the study accurately, several materials were used in the lab to make sure that the lab result was accurate. The materials that were used were reagents such as pH, enzyme concentration and acids. The tubes, notebook, pens and computers were used to conduct the study. The computer or laptop was used in the final phase to record and conduct tabulation of the result. The study was conducted in the biology laboratory where all materials need could be found.

Procedures

Enzyme Reaction

First, the tubes were set, and then the reagents were poured inside each tube. The tubes were set with Water, pyrophosphatase 3.0 ml and enzyme pyrophosphate. The enzyme of 2.5 ml was then added to each tube containing water, pyrophosphatase and triethanolamine buffer TEA, pH. The observation was made, and it was evident that after 30-minute intervals, the reaction of enzymes with the reagents started. Exactly after 2 minutes, the stopping agent was added to each reaction, including tubes where the enzyme was not placed. Immediately after 10 minutes, the tubes were transferred to room temperature for the color to develop. The observation was done and the reaction note for further analysis.

Effect Of Enzymes Concentration On Hydrolysis Reaction

The study discovered that the rate of enzyme reactions is affected by the enzyme concentration, and therefore, the higher the concentration of enzyme, the higher the rate of reactions. It is evident the hydrolysis reactions are higher when the concentration of enzymes is higher. The result indicates that when the rate of enzymes is 0 ml, the rate of hydrolysis reaction is 0.021 min/mL. The reaction increases as the concentration of enzymes increases along the graph. This is because when the concentration of enzyme increases, the substrate concentration increases as well, and therefore, the rate of reaction increases (Glatter O & Esterbauer, 2015, p. 23).

Based on the result in Figure, at 0ml concentration of enzyme, the substrate is not available, and therefore, the reaction will occur, but a product will be formed. Therefore, the reaction is lower or not there. In the process of enzyme reaction, a substrate is needed so that it catalyzes the process of reaction. The substrate is a substance that molecules use to act upon so that they can activate their reaction. According to Glatter and Esterbauser (2015, p. 15), when the reaction has less substrate, the reaction will decline; this is because there will be no substrate to react with the enzyme, and therefore, the reaction between substrate and enzyme will form a product hence the hydrolysis reaction will decrease. It is, therefore, evident from the result above in (Fig: 2) that the higher the concentration of enzyme, the more the hydrolysis reaction. It can be concluded that the concentration of enzymes enhances or increases the hydrolysis reaction.

Effect Of PH

The result obtained indicates that pH affects the reaction, and therefore, it affects the process of hydrolysis. It shows that the higher the pH, the higher the rate of hydrolysis reaction. From the reaction of pH and enzymes, it is recorded that the lower the pH, the lower the rate of reaction. The result in p, the PH reaction noted in the graph, indicates that when the PH level is 4, the average rate of hydrolysis reaction is 0.02mL/min. When the pH level increases to 10.5, the average rate of hydrolysis reaction increases to 0.041 mL/min. Therefore, it is evident that pH level affects the rate of enzyme reaction. This is because a higher level of pH causes more collision of enzymes and, therefore, increases the rate of enzyme reaction. The collision of enzymes makes it possible for the substrate to bind to the enzyme’s active position repeatedly, hence increasing the rate of reaction. It is, therefore, that the level of pH affects the enzyme reaction and hence increases or decreases the rate of hydrolysis reaction.

Effect Of Temperature

From the result obtained, it is evident that temperature affects enzyme reaction. The higher the temperature, the higher the average rate of reaction of the enzyme, and therefore, an increased increase requires a higher temperature. Since the enzyme has a maximum temperature at which it works best. The temperature above the optimum, the reaction of the enzyme will automatically reduce. In the graph (figure 4), the optimum temperature for the enzyme reaction is 70 degrees, and after 71 degrees, the enzymes become inactive, and the reaction will not occur; therefore, the reaction decreases tremendously. It is because for enzyme reactions to occur, the temperature is needed to allow more collision to take place, and therefore, it will make substrate tind with enzymes and therefore, more reaction will take place. The more binding between substrates and enzymes increases, the more reaction, and therefore, the average rate of hydrolysis reactions will increase.

The result obtained from the experiment is consistence with the expected result since other studies and literature have shown that the rate at which the enzyme reacts is affected by temperature, pH level and the concentration level of the enzyme. According to Temelli (2014, p. 24), the reaction of enzymes depends on the number of substrates inside the reaction and when the concentration of enzyme is higher substrate substance is many, and therefore, it increases the rate of reaction. Research has also established that the rate of chemical reaction is influenced by the enzymes, and the higher the concentration of enzyme, the higher the chemical reaction (Temelli, 2014, p. 23). However, the result obtained from the experiment is agreed or similar to other studies that have been conducted earlier.

However, the error could have been realized when reagents were being mixed, could be realized during tabulation because humans can register errors when doing tabulation. It is also possible to have errors when the reagents or tubearere placed under room temperature. Anything might have mixed with the test which cannot be identified easily. The improvement can be done to avoid any chances of making an error in the final result, such as monitoring the result continuously.

Conclusion

Based on the result obtained from the study conducted in the lab, it is evident the concentration of enzyme affects the rate of enzyme reaction. This result agrees with other studies which have been conducted by different scientists. The result of the study shows that when the concentration of the enzyme is 0mlL, the rate of hydrolysis reaction is 0.02mL/min. From the result of the experiment conducted, it can be summarized that the level of concentration of enzyme influences the chemical reaction. The result of the effect of pH is evident from the result obtained.

The result indicates that the lower the pH level, the lower the rate of enzyme reaction. It, therefore, can be concluded that the level of pH affects the reaction of enzymes and, therefore, influences the chemical reaction. Based on the experiment conducted on the effect of temperature on enzyme reaction, the result indicates that the temperature level affects the rate of the enzyme reaction and the higher the temperature, the higher the average rate of enzyme reaction. It can, therefore, be concluded that temperature level influences the rate of chemical reaction.

Bibliography

Glatter O, S., & Esterbauer, S. (2015). The effect of enzyme concentration on the rate of the hydrolysis of cellulose. 12 (4), 2-45.

PYNES, G. D., & YOUNATHAN$, E. (2012). Purification and Some Properties of Inorganic Pyrophosphatase from Human Erythrocytes. The JOURNAL OF BIOLOGICAL CHEMISTRY, 2-38.

Temelli, R. J. (2014). Effects of water on enzyme performance with an emphasis on the reactions in supercritical fluids. Journal of Science in Biology, 2-45.