Introduction Enzymes are the ultimate catalysts of living things. Enzymes are made of proteins which are structured and directed by amino acids chains. Enzymes attract and fit substrate molecules to an active site. The active site binds the substrate molecules covalently to enzyme forming an enzyme-substrate complex, which catalyzes the substrate molecule into a product. Enzymes have the capability to break or build compounds which keep cell systems functioning. For example, our digestive tract has catabolic enzymes which break apart food for storage and reuse. Our lab experiment studied the relationship of benzoquinone production when altering the pH and temperature of the reaction, as well as enzyme and substrate concentrations. We used a Spec 20 to evaluate absorbance at 540 nm. Absorbance represents rate of product formation. We tested the substrate catechol and the enzyme catacholase and recorded the product benzoquinone.
The objective was to identify the optimal conditions for the enzyme catacholase. I predicted that the reaction rate will rise with increasing enzyme concentrations until an optimal reaction rate is attained. This is because there are only so many substrates that need to react with the active sites on the enzymes. Once this equilibrium is reached the graph will stabilize and remain constant. I predicted that the reaction rate will increase with increasing substrate concentrations until an optimal point where there aren’t enough active sites on the enzymes to accommodate the influx of substrate molecules.
At this point the graph will reach equilibrium and remain constant. I predicted that heat would accelerate the reaction because if molecules have high kinetic energy their motions will become increasingly rapid. An incline in the graph will be evident but a decline will also occur because proteins denature at high temperatures. The enzyme will lose its shape and thus its work function will diminish, like an overheated car. This graph should resemble a pyramid. I predicted that the most acidic and basic environments would have a low level of reaction and that neutral pH will produce the greatest level of reaction. Living systems operate in a certain cell environment, pH levels vary in hydrogen ion concentrations. I presumed that the potato would need a neutral pH solvent because most enzymes generally function at neutral pH levels. This graph will resemble a bell curve. Methods
We conducted four experiments. All four test results measured benzoquinone production by a spectrophotometer at 540nm. Higher levels of absorbency means higher rates of benzoquinone production. In the first experiment we tested how enzymatic concentration will affect the level of benzoquinone production. We had four treatments all; with equal total volume, a constant substrate concentrations and varying enzyme extract concentrations (Table A.1). The enzyme-substrate complex will form and react spontaneously upon adding the extract; therefore in all experiments catacholase was added last to each test tube. We covered the four tubes with parafilm and inverted the tubes for three minutes at one minute intervals. We then recorded the absorbance.
In activity B our objective is to determine if substrate concentration will affect the level of benzoquinone production. There are six treatments each have equal total volume, constant extract (enzyme) concentrations and varying substrate (catechol) concentrations (Table B.1). After amounted water and substrate is mixed the enzyme was added last. The tubes were covered with parafilm and inverted for three minutes at a one minute intervals. Once each tube has been processed for three minutes re-zero the Spec and then record the absorbance units.
In activity C our objective is to test how different temperatures will affect the rate of benzoquinone production. In our four treatments each was held at a certain temperature (Table C.1). We prepared the temperate environments by; putting 100 mL of tap water with boiling chips in a beaker and on a hot plate, we placed ice into a separate beaker for an ice bath, we positioned a tube rack for room temperature test, and then placed another beaker into a warm water bath. We measured 4 mL of catechol and 1 mL of water into all four tubes. After we covered the four tubes with parafilm and inverted followed by the incubation of tubes in their designated environments for five minutes. After five minutes we added 1 mL of potato extract (enzyme) and covered all but the boiling tube. We inverted the catacholase (to mix boiling tube we carefully stirred). After adding the enzyme we measured the temperature of each tube and then incubated them in their temperate environments for five more minutes. Following five minutes we prepared the Spec and recorded the absorbance.
In activity D our objective was to test whether pH level would increase or decrease benzoquinone production. We added varying pH levels into four tubes based on (Table D.1). We covered the tubes with parafilm and inverted each for three minutes in one minute intervals. After three minutes we prepared the Spec and recorded each tube’s absorbance. Results and Discussion
As the enzyme concentration increased, there was a solid increase in benzoquinone production (Figure A.1). My hypothesis stated that product formation would rise until a maximum was obtained. Based on the data, my results did not support my hypothesis. From this experiment I learned that the concentration of enzymes can significantly speed up reaction rate and that enzymes are versatile in that they will bind substrate molecules and catalyze at a high rates even if the substrate level is constant. This test could be improved by including more enzyme concentrations to broaden our data spectrum and perhaps then my hypothesis will be supported. As substrate concentration increased, there was an increase in absorbance followed by a plateau around 1.6 ml of catechol (Figure B.1).
My hypothesis stated that product formation would rise until a maximum when absorbance would plateau because substrate molecules would outnumber enzyme active sites. Based on data gathered, results support my hypothesis because with 0.5 :1 substrate to enzyme ratio, production began to level off. From this experiment I learned that catechol concentration has a big impact because the substrate represents the workload that needs to be acted on and therefore the higher concentrations can overload enzyme workspace. If we were to expand this experiment and include more substrate concentrations then we could assess the hypothesis more accurately to see if the enzyme reaction rate continues to remain constant when enzyme occupation is reached. Benzoquinone product formation climaxed at 52 degrees Celsius this is the optimal functioning rate, any temperature to follow will obstruct the enzyme’s function and result in decreasing levels of product formation (Figure C.1).
My hypothesis stated that the enzyme will function faster with activation energy supplied from kinetic heat. But that this fast rise will subsequently be prone to a rapid decrease because once optimal temperature is breached, the tertiary structure of the enzymatic proteins will unravel into dormant amino acid chains. Our test results support my hypothesis in that there is a cone shaped graph which represents a maximum functioning level of 52 degrees Celsius. There is a rapid decline at 99.3 degrees Celsius. From this experiment we learn that enzymatic rate of product formation depends on the solution temperature. Such as the maladies fever instills, high temperature denatures proteins which disable enzymes capability to function properly. We could expand this experiment by testing different substrates other than catechol to see if this temperature relationship/pattern is applicable to other enzymatic reactions. The high acidic and basic solvents had a low reaction rate while the neutral/basic environment had the highest benzoquinone production (Figure D.1).
My hypothesis stated that the graph would resemble a bell curve because most enzymes catalyze within a neutral solvent environment. Our test results support this hypothesis slightly because there was a bell shape curve, however I learned that the enzyme catacholase generates benzoquinone faster in a slightly basic environment specifically pH 8. We can expand this experiment by using other substrates (not catechol) with the same test process we can compare what kinds of substrates operate in certain pH environments. It would be interesting to find a pattern or categories in which substrate react accordingly with, for example the enzymes in our gut work in a slightly acidic microenvironment. Conclusion
Enzymes help biological systems function. They induce the biochemical process by holding reactants in position and catalyzing the reaction to form the product. Catecholase, the enzyme, lowers the activation energy needed for benzoquinone to form, which makes the chemical reaction happen much quicker. The rate of enzymatic reaction is very important to every organism’s survival. We act accordingly to ensure a stable environment in our bodies so that our metabolism and immune systems function properly. The enzyme is like a crane that transfers iron rods onto a building structure, without that mechanism the building would take forever to build! Multiple chemical reactions occur for a process to function. If the enzymes which catalyze such reactions aren’t regulated, metabolic pathways would result in chaos. The objective in this experiment was to evaluate what solute concentrations and physical environments the enzyme catacholase would function best to.
We tested what enzyme and substrate concentrations and what temperature and pH levels would generate the highest reaction rates. We analyzed how these variables would effect enzyme function by measuring the rate of benzoquinone production in absorbance. We found that high enzyme concentration would increase product formation; that high substrate concentrations would plateau when no enzyme work sites are left vacant; that catacholase denatures after the optimal heat of 52 degrees Celsius; and that catacholase functions best at a pH 8 level. It is important to understand how enzymes perform the best because then we can have greater control over the maintenance of these vital systems.
For example your body can become too acidic from alcohol poisoning which causes body malfunctions and ultimately a black out. In this lab I found that the enzyme catacholase worked best when enzyme concentration was greatest resulting in a positive linear graph, and that the substrate concentration would as well enhance reaction rate until 1.5mL of catechol concentration, at which point enzyme reactors are all occupied and the graph plateaus. We also can conclude that 52 degrees Celsius is catacholase’s optimal function level and that a slightly basic pH solvent is most efficent for product formation.
These experiments represent a selective study based on limited solute concentrations and temperature and pH test environments. Further study on enzymatic functions can proceed by adding more substrate and enzyme concentrations, this will expand data and grant a bigger picture as to how reaction formation will continue. In relation to enzymatic environment levels we can further the temperature and pH experiments by incorporating a different solute unlike catachol to determine if any patterns of favored microenvironments develop.
Photography and fact discussion
Photography is often associated with factual reporting. The camera, however, records only one point of view, and that can be edited quite easily today. Photographers choose which pictures to take and which to ignore. Editors select only a few images to print. Governments can control the press’s access to events and thus influence coverage, as happened in the early 1990s during Operation Desert Storm. Is the news factual? Is art factual? Cite some examples to support your argument.