Enzymes Are Highly Selective and Substrate-Specific Catalysts Lab Report

Enzymes are highly selective and substrate-specific catalysts that work by lowering activation energy for reactions thus increasing the rate of metabolic reactions. In enzymatic reactions, substrates are molecules binding onto enzymes' active sites to form enzyme-substrate complexes (Cornish-Bowden, 2004). Lactose is a disaccharide sugar commonly found in milk and lactase is the enzyme responsible for catalyzing lactose into its subsequent monosaccharide products; glucose and galactose. In line with this, lactose intolerance is the inability to digest lactose; lactose intolerant individuals have insufficient levels of lactase and symptoms include flatulence, diarrhea, rumbling stomach, and vomiting as well (Wilson, 2005).

There are several factors that affect enzymatic reactions. According to Dunaway-Mariano (2008), enzymatic activities are affected by temperature, pressure, chemical environment such as pH, and substrate concentration as well (Dunaway-Mariano, 2008). To determine the optimal conditions for enzymatic activity, the enzyme, lactase was tested under four conditions; different temperatures, pH, substrates, and with/without cofactors.

For the laboratory experiments, it was hypothesized that lactase works best at temperatures of 40 degrees Celsius which is closest to a human's body temperature. In addition, if the lactase used in this experiment was extracted from human cells, it is assumed to work best at pH of about 6 and 7; slightly acidic pH. Moreover, lactase should be specific to lactose and the addition of chelating factors such as EDTA to lactase-mediated reaction takes away some co-factors required for effective functioning of the enzyme making the reaction slower.

Specificity is the affinity which an enzyme has for its substrate. The substrate is flexible and fastens to the active site of the enzyme where the catalysis of substrate's reaction occurs. The active site normalizes by returning to its original shape when substrates detached and have formed products. The enzymatic active site comprises of amino acids and amino acid side chains. It is expected that the amino acid side chains chemically interact with the enzymatic substrate. Therefore, the enzymatic substrate specificity is revealed by the active site, an indication that enzymes should better catalyze and hold to its substrate than for others. Since enzymes have unique structure, it is very specific to the substrate it can catalyze. Each enzyme has its own specific substrate or set of substrates not any substrate can be a general active site of the enzyme.

Cofactors are non-protein compounds (usually metal ions) which bind with enzymes to initiate the enzyme's catalytic reaction. Cofactors vital for enzyme catalytic reactions include iron, copper, and magnesium as well as potassium among others. However, the removal of a cofactor from the enzyme's structure results in the loss of its catalytic activity. In line with this, coenzyme is used to describe cofactors participating with the enzyme in catalytic reactions.

Material and Methods

The Effect of Temperature on Enzymatic Activity

Six microfuge tubes with temperatures of 0, 25, 40, 60, 80, or 100 were filled halfway with lactase solution of 500 µL using a plastic pipette. After filling the tubes with the solution, the tubes were placed in beakers with heated water matching each temperature for five minutes. While still in the beakers, milk was added to each tube until lactase and water mixture reached full; this was 1 mL of mixture in the tubes. Immediately after the ten minutes, a glucose strip was placed in each tube for one second, and then removed and allowed to sit on the bench-top for thirty seconds. After the thirty seconds, the colorations of the strips were compared to the chart provided to determine the amount of glucose in mg/dL. The values were then recorded in a table.

The Effect of pH on Enzymatic Activity

Six microfuge tubes 2, 4, 6, 7, 10, and 12 were filled halfway using a plastic pipette with the appropriate pH buffer. Afterwards, three drops of milk was added to the tubes using a clean plastic pipette. The mixture was then shaken to completely mix the milk and pH buffer. Thereafter, by using a clean plastic pipette, three drops of lactase solution was added to each tube. This mixture was then shaken gently to completely mix these substances. When a uniform mixture had been obtained, the tubes were placed in a 40° C. water bath and incubated for approximately 10 minutes. After the 10 minutes, a glucose strip was placed in each tube for one second, then removed and left to sit on the bench top for thirty seconds.At the end of thirty seconds, the colorations of the strips were compared to the chart provided to determine the amount of glucose in mg/dL.

Enzymatic Specificity

Two microfuge tubes labeled L; for lactose and M; for maltose were filled halfway with equal amount of lactose and maltose using a clean pipette. After this, by using a clean plastic pipette, lactase solution was added to each tube, until the level of mixture in each tube was full; with 500 µL of lactase and 500 µL of maltose. Thereafter, the tubes were placed in a 40°C water bath and incubated for 10 minutes. At the end of ten minutes, a glucose strip was dipped in each tube for one second, then removed and allowed to sit on the bench top for thirty At the end of thirty seconds, the colorations of the strips were compared to determine the amount of glucose in mg/dL.

Determining Cofactors of Enzymes

Two microfuge tubes one labeled "Control" and another one "EDTA" was filled a-quarter with distilled water and EDTA respectively. Thereafter, three drops of milk were added to each tube, and the tube inverted and allowed to sit for 1 minute. After inverting and sitting the tubes for one minute, they were placed in a 40° C. water bath and left to stand for approximately 10 minutes. After 10 minutes, a glucose strip was placed in each tube for one second, and then removed and allowed to sit on the bench top for thirty At the end of thirty seconds the coloration of strips were compared to the chart provided. The amounts of produced glucose in mg/dL were recorded in the table below.

Results and Discussions

Results

0° C

Glucose (mg/dL)

25° C

Glucose (mg/dL)

40° C

Glucose (mg/dL)

60° C

Glucose (mg/dL)

80° C

Glucose (mg/dL)

100° C

Glucose (mg/dL)

10

Minutes

0

Table1: Enzymatic Activity of Lactase at varying temperatures based on Glucose production

As seen from the above results, 250 mg/dl of glucose was produced at 0 degrees Celsius in a span of 10 minutes. At 25 degrees Celsius, 500 mg/dl was produced in 10 minutes. The production of glucose peaked at 40 degrees Celsius where it was 1000mg/dl in 10 minutes and remained constant at 60 degrees Celsius. At 80 degrees Celsius, the production went down to 280mg/dl in 10 minute and further to 0 at 100 degrees Celsius.

pH

2

4

6

7

10

12

Glucose (mg/dL)

Mg/dl

Mg/dl

Mg/dl

Mg/dl

0

Mg/dl

0

Mg/dl

Table2: Enzymatic Activity of Lactase at varying pH based on Glucose production

As observed in the above table, the production of glucose was 500mg/dl at 2 pH level and remains constant through pH level 7, but at 10 the production plummeted to 0 through pH level 12.

After 10 minutes

Lactose Tube (glucose mg/dL)

Maltose Tube (glucose mg/dL)

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

2000

Table3: Determining the specificity of Lactase in the presence of Lactose or Maltose

In determining the specificity of lactase in the presence of lactose or Maltose, the first group had 500mg/dl of glucose in the lactose tube and 1000mg/dl in maltose tube. Group 2 had 1000mg/dl of glucose in both the tubes and the same was for group 3 and 4 except for group 5 where glucose was 1000mg/dl in lactose tube and 500mg/dl in maltose tube. In group 6, lactose tube recorded 2000mg/dl of glucose and 500mg/dl in maltose.

Control-Glucose (mg/dL)

EDTA-Glucose (mg/dL)

0

0

0

0

0

Table4: Utilizing the chelating agent EDTA to determine if Lactase activity is dependent upon a cofactor

As seen above, there was a variation of the amount of control glucose ranging from 250mg/dl to 500mg/dl in all the instances except in one where EDTA glucose was recorded at 100mg/dl against control of 500mg/dl.

Discussion

Temperature is one factor which affects enzymatic activities as seen in table 1 above. As the temperature increased, the rate at which enzyme lactase hydrolyzed the lactose into glucose and galactose increased, and did not seem to slow down until 40 degrees Celsius. After reaching 40 degrees, the reaction stabilized and started lowering after 60 degrees Centigrade. From this observation, it is clear that temperatures between 0 to 25 degrees were too cold and slowed activity of enzyme lactase. Therefore, the rising temperature resulted in faster rate of reaction due to the increasing.....