Enzymes

Summary:   A study of the effects of differnt enzymes on the rates of reaction. These effects are created through an experiment using the enzyme acid phosphates (ACP) and the substrate p-nitrophenyl phosphate.

---

Introduction:

Enzymes are an important part of all metabolic reactions in the body. They are catalytic proteins, able to increase the rate of a reaction, without being consumed in the process of doing so (Campbell 96). This allows the enzyme to be used again in another reaction. Enzymes speed up reactions by lowering the activation energy, the energy needed to break the chemical bonds between reactants allowing them to combine with other substances and form products (Campbell 100). In this experiment the enzyme used was acid phosphates (ACP), and the substrate was p-nitrophenyl phosphate.Enzymes are very specific in nature, which helps them in reactions. When an enzyme recognizes its specific substrate, the enzyme binds to the substrate in a region called the active site which is made of amino acids. Once the substrate binds, the enzyme changes its shape slightly to make an even tighter fit around the substrate, This is called induced fit and it allows for the enzyme to catalyze the reaction more easily. Another factor contributing to catalyses is the amount of substrate present; the more substrate molecules available, the more often they bind the active site. Once all of the enzyme's active sites are occupied by substrate, the enzyme is saturated ( Campbell 99). Enzyme's have optimal conditions under which they perform. These include temperature, pH, and salt concentration, amongst others. In this lab we only focused on pH and temperature. Each enzyme is specific to a certain optimal temperature and pH. When conditions are favorable, the reaction takes place at a faster rate, allowing for more substrates to collide with active sites of enzymes. However, if conditions get too extreme, the enzyme will denature, or become inactive due to a conformational change in shape (Campbell 100). Substances known as inhibitors can also stop an enzymatic reaction by either directly binding to the active site or by binding to the enzyme at a different site on the enzyme which changes the shape of the enzyme. In this experiment, NaOH was the inhibitor used to stop the enzymatic reactions. NaOH is very basic and when added to a solution, will cause a drastic increase in pH, causing denaturation of the enzyme. The amount of product formed could be calculated by placing the test tube in a spectrometer after the addition on NaOH. A spectrometer measures the absorbance of a solution, which helps compare how much of a substance is in a solution.

I hypothesized that the rate of the reaction would increase, producing more product as the amount of ACP in solution was increased because more enzymes allow for more substrate to be converted to product. The same hypothesis was made that when we increased the substrate, p-nitrophenyl phosphate, the amount of product produced would increase as well because there would be more substrate that could bind to the enzyme and be converted to product. For the environmental experiments, both temperature and pH, I predicted that the amount of product formed would increase with the temperature and pH, but then begin to decline after the enzymes reached optimal conditions. In other words, at the optimal temperature and pH, the enzyme velocity would be greatest, producing the most p-nitrophenol. Also, I predicted when the pH and temperature became too extreme, the enzyme would no longer be reactive due to denaturation. Overall, enzymes are very specific proteins that play a key role in metabolic processes and like all other proteins have optimal conditions under which they function most successfully.

Materials and Methods:

For a complete list of the materials and methods used in this lab please reference the laboratory manual (Lombard and Terry 92-103).

Results:

As seen in figure 1, as the amount of p-nitrophenol increases, the absorbance of the solution increases as well. The amount of p-nitrophenol that gives the greatest absorbance of 2.250, is .375micromoles. Oppositely, with no p-nitrophenol, the absorbance is 0.

Figure 1

Figure 2 shows a general increasing trend in production of p-nitrophenol produced as the amount of enzyme, ACP was increased. The amount of p-nitrophenol was greatest when the amount of ACP in solution was greatest, 1.00milliliters. Oppositely, with 0.00milliliters of ACP, there was no production of p-nitrophenol.

Figure 2

Figure 3 shows the effect of substrate concentration on the production of a product. Similarly to figure 2, the graph shows an increasing trend in product production with the amount of substrate, p-nitrophenyl phosphate. The amount of p-nitrophenol produced was seven times greater when there was 1.00milliliters of substrate compared to when there was 0.0millilters.

Figure 3

Figure 4 shows an increasing amount of product formed as pH increases, but then begins to decline after a certain pH. The amount of p-nitrophenol produced was the least, .003micromoles, at a pH of 9 and greatest, .138, at a pH of 5. After a pH of 5, the amount began to decrease. Before a pH of 5, the amount increased.

Figure 4

Figure 5 shows the same general trends as figure 4. As the temperature is increased, the amount of product increases and then begins to decline after a certain temperature. The temperature that formed the greatest amount of p-nitrophenol was 50 degrees Celsius, producing .40micromoles. The temperature that produced the lowest amount was 100 degrees Celsius, giving only .004micromoles.

Figure 5

Discussion:

The overall results of the experiment showed the correct general trends, in regards to the effects of environmental and chemical factors on ACP. As expected, the absorbance of the solution increased as the amount of p-nitrophenol was increased. This makes sense because what the spectrometer is reading is the amount of p-nitrophenol ( the yellow color of the solutions). So therefore, is there is more p-nitrophenol added to the solution, there will be a greater absorbance.

Changing the enzyme concentration affects the amount of product formed, as seen in Graph 2, and Table 2. Previously it was hypothesized that as the amount of enzyme was increased, the amount of product formed would increase as well, and the results fully support this. With only .05 milliliters of ACP present, the amount of p-nitrophenol produced was only .04micromoles. Oppositely, the solution with 1.00 milliliters of ACP produced .357 micromoles, almost nine times as much. This makes complete sense, because with more enzyme in the solution, that means more active sites were available for the binding of substrates, allowing for more product to be produced. In contrast, if the amount of enzyme was decreased, the opposite would be expected to occur since there would be less active sites available. Similarly, increasing the amount of substrate will also increase the rate of the reaction, because "the more substrate molecules available, the more frequently they access the active sites of the enzyme molecules. (Campbell 99)" As seen in Graph 3, Table 3, values went from .002micromoles of p-nitrophenol to .128micromoles of product as the amount of substrate, p-nitrophenyl phosphate, was

increased from 0.0ml and 1.0ml. The results fully confirmed my hypothesis that an increase in substrate would produce an increase in product.

As stated earlier, every enzyme has an optimal pH value at which it works the best. Looking at Graph 4 and Table 4 the results for the pH experiment supported my hypothesis that the amount of p-nitrophenol produced would increase with the pH until it reached the optimal pH. It would then begin to decline as the pH value continued to increase and migrated away from the optimal pH. From the results, one can see that ACP worked best when placed in a solution with a pH of 5, this being the enzymes optimal pH value. When placed in solutions with pHs of 3 and 9, the amount of p-nitrophenol produced was lower than when placed in solutions with pHs of 4 and 7. This is due to the fact these pHs are closer to the optimal pH than the other two. This makes sense because the further away the pH is from optimal conditions, the reactivity of the ACP will decline, inhibiting the production of p-nitrophenol. That is why the solution with a pH of 4, the closest pH to optimal conditions produced .054 micromoles of p-nitrophenol and the solution with the pH of 9, the farthest away from optimal conditions, produced only .003 micromoles of p-nitrophenol.

The same concept applies for the experiment involving different temperature. As with pH, every enzyme has an optimal temperature. Both Graph 5 and Table 5 favored my hypothesis that the enzyme would work the best at an optimal temperature, but as the temperature moved away from optimal conditions, both increasingly and decreasingly, the enzyme activity would decrease causing the amount of p-nitrophenol to decrease. Looking at figure 4, one can see that the ideal temperature for ACP was 50 degrees Celsius because the amount of p-nitrophenol produced was .4umoles, almost three times greater than any of the other temperatures. This is because at the optimal temperature, the substrates collide with the active sites of the enzymes more often, causing more product to be produced (Campbell 100). The velocity of the reaction is at its maximum. At 100 degrees Celsius, almost no p-nitrophenol was produced. This is due to the fact that as the temperature continues to increase past the optimal, the hydrogen bonds that stabilize the active shape of the enzymes break, causing the enzyme to convert to an inactive shape. This is known as denaturation (Campbell 100). This can further be explained by the extreme specificity of the enzyme.

Although the results supported my hypothesis about the general trends of the experiments, the actual data was not exactly what was expected. As with any experiment, there was a number of human errors that could have contributed to the faulty data. The pippetors that were used were not exactly the best measuring devices. It was hard to get the exact amounts of liquids such as volume of ACP and its substrate p-nitrophenyl phosphate. This would have effected the reaction and the amount of product formed. Also, every time a test tube was placed in the spectrometer, we did not remember to wipe it with a Kim wipe before placing it in. This along with the fact that the spectrometers could have become unbalanced after using the blank tube, would have given faulty absorbance readings. These readings when examined with the standard curve, would led us to believe there was more or less product produced, than the actual amount. However, the biggest contributor to error was the addition of NaOH. When this was added to the solution, the reactions in the test tube stopped, so therefore no more product could be formed. We were supposed to add NaOH exactly five minutes after the reaction started. In most cases we either added it too early or too late. If it was added too late, the amount of p-nitrophenol produced would be greater since the reaction was given more time to occur. Oppositely, if added too early, the amount of product would be less than expected since the reaction was not given enough time to proceed. There was a general trend in the faulty data readings. The absorbance readings for test tube 5, were always further away from the expected values than test tube 1. This is because the NaOH was not added to each tube at a time, but in sequential order with the test tube numbers. This allowed the reaction in test tube 5 to proceed longer than in test tube 1, allowing more product to be produced, giving a higher absorbance reading than expected. In fact, this trend was shown in all the test tubes. In increasing order of test tube numbers, every absorbance was more off than expected.

I would change a few things about this lab. Firstly, I would have used a micropipette instead of the ones that we used because they would give more precise measurements. Also, I would have had five people in each group so that everyone could add NaOH to the solutions at the same time, stopping the reactions simultaneously.