Lab 1: Enzymes Table 1) Enzyme Succinic Dehydrogenase Utilizing Substrate Coupling of Succinic Acid and Methylene Blue to form

Fumaric Acid and Methylene White while being Competitively Inhibited by Malonic Acid (Data source: Steven Keirstead, Summer 2000)
Tube Succinic Methylene Deionized Number Acid Blue H2O 1 90 L 210 L 270 L Other 500 L Sucrose Calf Heart 0 UL Initial Color Final Color Enzyme Activity N/A No Activity No Activity

Blue Blue Transparent Transparent Tan Blue Opaque Blue Opaque Tan Blue Opaque

90 L

0 L

480 L

500 L

0 L

210 L

360 L

500 L

90 L

210 L

270 L

500 L

90 L

210 L

240 L

30 L Malonic 500 L Acid

Blue Opaque

330 L

210 L

0 L

30 L Malonic 500 L Acid

Blue Opaque

Small bits of blue on top, Uninhibited nearly clear on top of tan Dark blue green on top Very of a small Inhibited amount of tan Small section of Slightly blue green Inhibited on top, most of tube tan

Table 1. Tubes 1-3 function as negative controls while tube 4 functions as a positive control. Test tube 5 shows what happens when an inhibitor is added and test tube 6 shows what happens when both an inhibitor and additional substrate are added. Comparing test tubes 5 and 6 it can be seen that Malonic Acid is a competitive inhibitor, as when we add more substrate we can overcome the effects of inhibition. 1. The enzyme succinic dehydrogenase uses coupled reactions and needs both substrates, succinic acid and methylene blue, to create products. When the enzyme is active succinic acid will become oxidized to fumaric acid and methylene blue will become reduced to methylene white. As methylene blue becomes reduced it changes colors. The enzyme is active when there is a color change. 2. Calf heart extract acts as our enzyme and will catalyze the reactions. Sucrose is a negative control and neither inhibits nor catalyzes the reactions. Mineral oil prevents the re-oxidation of methylene white back to methylene blue by oxygen from the air.

3. In tubes 1-3 there was no enzyme activity and therefore no color change. In tube 4 there was uninhibited enzyme activity and therefore a drastic color change from blue to nearly all tan. In tube 5 there was inhibited enzyme activity so only a very slight color change from blue to blue on top of tan. In tube 6 there was slightly inhibited enzyme activity so there was a large color change from blue to tan with bits of blue on top. The more the tube color changed the higher the enzyme activity was. 4. Tube 1 is a negative control as it is missing the enzyme. Tube 2 is a negative control because it does not contain methylene blue. Tube 3 is a negative control because there is no substrate present. Tube 4 is a positive control as it contains all the components, but the experimental component (inhibitor). Tubes 1-3 show what happens when we are missing a certain component. Tube 4 shows what happens when we have all the components. Comparing tube 5 to tube 4 we see what happens when we add an inhibitor. Comparing tube 6 to tubes 5 and 4 we see what happens when we add both an inhibitor and more substrate. 5. Competitive inhibition can be overcome by the addition of more substrate therefore with more substrate added tube 6 should have a large color change. Noncompetitive inhibition cannot be overcome by adding more substrate. Adding more substrate to tube 6 would not have a large influence on color change the color of tube 6 would remain almost the same. 6. As there is a large color change in tube 6 the enzyme is competitively inhibited. In the presence of more substrate, the substrate and enzyme are able to bind at a high rate as if there werent any inhibitor present. 7. The structure of malonic acid and succinic acid are quite similar. This suggests that malonic acid binds to the active site, which is consistent with the answer to number 5 of competitive inhibition.

Figure 1) Effect of -Amylase concentration on the reaction rate of the hydrolysis of amylose starch (Data source: Jordan Roberts and Jessica Leung, September 2010)
0.180 0.160 Reaction Rate (mg/sec) 0.140 0.120 0.100 0.080 0.060 0.040 0.020 0.000 0% 2% 4% 6% 8% Enzyme Concentration (%) 10% 12% y = 1.5533x + 0.0095 R = 0.9977

Figure 1. There is a positive correlation that is linear between the enzyme concentration and reaction rate. As the solution becomes more concentrated with the enzyme -Amylase the rate at which amylose starch is broken down increases in a linear fashion. 1. Hypothesis: If more -Amylase enzyme is present then the time it takes the amylose starch to hydrolyze will decrease. 2. The trend in the above data supports the hypothesis from question 1. Looking at Figure 1 it can be seen that as the enzyme concentration increases so does the reaction rate. With more -Amylase (enzyme) available, indicated by the higher concentration, amylose starch (substrate) is able to bump into Amylase more frequently and therefore hydrolyze faster in a shorter time period and increase the reaction rate. 3. Enzyme concentration is the limiting factor. By increasing enzyme concentration the reaction rate increased. This indicates that enzyme concentration is what controls the reaction rate.

Figure 2) Effects of pH on activity of enzyme -Amylase in hydrolyzing amylose starch (Data Source: Noel Ayoub and Dan Trafficonte, Summer 2007)
0.250

0.200 Reaction Rate (mg/sec)

0.150

0.100

0.050

0.000 0 1 2 3 4 5 pH 6 7 8 9 10

Figure 2. The enzyme -Amylase is most effective at a pH of 7. At a pH of 7, the reaction rate is the highest. As the pH moves away from 7 the activity of the enzyme drops. 1. Hypothesis: If the pH of the solution is close to physiological pH (7.365) then the enzyme -Amylase will be very active. 2. This hypothesis is supported by the above data. Looking at Figure 2, it can be seen that the reaction rate is the highest around a pH of 7. This means that the enzyme is highly active in breaking down amylose starch at a pH of 7. 3. The charges at the enzymes active site influence the enzymes activity. If the charges at the active site are not correct then the enzyme will not be able to attract the substrate. The charges at the enzymes active site are affected by pH. Every enzyme has a pH in which that enzyme functions optimally. If the pH of the enzymes surrounding solution is not that optimal pH the enzyme will have the wrong charges and therefore low activity. 4. The approximate optimum pH for -Amylase is a pH of 7. 5. I would expect to find -Amylase in the small intestine. The small intestine has a pH of 6-7 whereas the stomach has a pH of approximately 2. As Amylase functions optimally at a pH of approximately 7 it would more likely be found in an area with that environment, the small intestine. In the stomach, Amylase would not be very effective.