Number 9

I. Objective(s)

The primary goals of the experiment are to ascertain how specific environmental factors
influence enzymatic reaction velocity through analysis of the kinetics of a β-fructofuranosidase
catalytic reaction. The factors to be tested include enzyme concentration, substrate concentration,
and pH values. The Michaelis constant (KM) and the maximum velocity (Vmax) of an enzymatic
system will be determined and the ways in which they are affected will also be assessed.

II. Theory/Background

Enzymes are molecules that facilitate reactions, but are not consumed in the process. In the
enzymatic catalysis of chemical reactions, the velocity of the reaction can be influenced by a
variety of environmental factors. The factors include the concentrations of enzymes and
substrates, the pH of the solution, the temperature, and whether or not crucial cofactors or
inhibitors are present in the system. The experiment concerns the enzyme β-fructofuranosidase,
which is the catalyst that assists in hydrolyzing sucrose. One notable aspect of sucrose hydrolysis
is that the original optical rotation, dextro, is altered to the levo form, which allows an equimolar
mixture of D-glucose and D-fructose, or invert sugar, to be formed. The production of invert
sugar allows sucrose hydrolysis to be monitored through the use of reducing sugar techniques. A
reaction with a reducing sugar yields a visible color change relative to the concentration of the
reducing sugar used, and the reaction can therefore be practical both quantitatively and
qualitatively. In order to draw conclusions from reducing sugar reactions, it is necessary to first
construct a standard curve that plots absorbance versus the micromoles of hydrolyzed sucrose.
Furthermore, in accordance with the Michaelis-Menten equation so what is the equation?, it is
expected that if the substrate concentration remains at a constant value that is not limiting, the
velocity of a chemical reaction catalyzed by an enzyme should be of the first order regarding
theconcentration of the enzyme. How about when keeping enzyme constant and varying the
substrate concentration? Also, enzymes possess an optimal pH, so when the velocity of the
enzymatic reaction is plotted against pH, a bell-shaped curve will be produced.

Moreover, additional important aspects of enzymatic systems involve calculating the values of
the Michaelis constant (KM) Define KM. and the maximum velocity (Vmax) as well as how
other substances influence those values. The Michaelis constant and the maximum velocity can
be assessed in the effects of the alternative substances on an enzymatic reaction. Various
inhibitors can display significant effects on such systems. For instance, competitive inhibitors
raise the KM value but do not alter the Vmax. On the other hand, non-competitive inhibitors
lower the Vmax and do not impact KM, whereas un-competitive inhibitors will have negative
effects on both values.

III. Materials

The materials and equipment to be utilized during the experiment include: 7.5 mL of glucose and
fructose standard, 27.5 mL of water, 56 mL of DNS reagent, a cuvette, a Spectronic 21, a boiling
water bath, a 37oC water bath, Microsoft Excel, 15.5 mL of β-fructofuranosidase in acetate
buffer, 35 mL of acetate buffer, 5 mL of unbuffered enzyme in water, 6 mL of buffer per whole-
number pH value from 3.0 to 7.0, and 7.5 mL of 0.1 M sucrose in acetate buffer and sucrose in
water.

IV. Procedures

The first major step in the experiment is to construct a standard curve that compares absorbance
versus the micromoles of hydrolyzed sucrose. Six tubes were prepared with a standard
containing 1 μmole of glucose and 1 μmole of fructose, with 1 mL of the standard being the
equivalent of 1 μmole of sucrose, and water. The tubes, numbered 0 through 5, were prepared
with the following ingredients and amounts in accordance with the lab protocol provided by Dr.
Huang (Table 1).

Table 1: Preparation of samples according to the lab protocol provided by Dr. Huang.

Tube Number mL of Glucose + Fructose Standard mL of Water

0 0 5.0
1 0.5 4.5
2 1.0 4.0
3 1.5 3.5
4 2.0 3.0
5 2.5 2.5
Each mL of the standard used consisted of a solution with 1 μmole of glucose and 1 μmole of
fructose, which was equivalent to 1 μmole of sucrose. Once all tubes were prepared, each tube
received 2 mL of DNS reagent, which was handled carefully due to its strong alkalinity. The
mixture was then completely blended and, with the water bath at a rolling boil, the tubes were
placed into the boiling water bath to heat for 5 minutes. The tubes were left to stand to permit
cooling and then the absorbance values for tubes 1 through 5 were taken at a wavelength of 540
nm with the Spectronic 21. Tube 0 was set at zero absorbance so it could be used as the blank.
Using Microsoft Excel, the absorbance values were plotted on the y-axis versus the micromoles
of sucrose equivalent to the standard on the x-axis and a line of best fit was superimposed on the
plot.

The next test assessed how the velocity of the enzymatic reaction was influenced by enzyme
concentration. Six test tubes were prepared in accordance with the following lab protocol
provided by Dr. Huang (Table 2).

Table 2: Preparation of samples according to the lab protocol provided by Dr. Huang.

Tube Number mL of Enzyme in Acetate Buffer: mL of Acetate Buffer

β-fructofuranosidase/invertase
0 0.0 4.0
1 0.5 3.5
2 1.0 3.0
3 1.5 2.5
4 2.0 2.0
5 2.5 1.5

A separate test tube 8 mL of a substrate solution composed of 0.1 M sucrose in acetate buffer
was grouped with the other 6 tubes and put into a hot water bath near 37°C for 5 to 10 minutes to
allow the temperatures of the solutions to match that of the bath. The reactions were carried out
so that each tube would react for a total of 10 minutes. The heated substrate was added in 1 mL
quantities to tubes 0 through 6 in 15-second increments. After 10 minutes following the addition
of the substrate to tube 0, the reaction was halted by the addition of the DNS reagent in 2 mL
quantities to tubes 0 through 6 in 15-second increments. All tubes were reheated in the boiling
water bath for an additional 5 minutes, and then the absorbances of each were taken at a
wavelength of 540 nm except for tube 0, which served as the blank with the absorbance set for
zero. The standard curve prepared prior to the test was used to calculate the number of
micromoles of hydrolyzed sucrose in 10 minutes and Data Table 1 was completed (see Results
section). A plot was constructed with velocity on the y-axis and mL of the enzyme used on the x-
axis, which was then compared to the standard curve.

The next test assessed how various pH values affect the velocity of an enzymatic reaction with β-
fructofuranosidase. Ten test tubes were prepared in accordance with the lab protocol provided by
Dr. Huang:

Table 3: Effect of pH on the velocity of an enzymatic reaction with β-fructofuranosidase.

Tube Number mL of Unbuffered mL of Water mL of Buffer

Enzyme in Water
1 1 0 3 mL at pH 3.0
2 1 0 3 mL at pH 4.0
3 1 0 3 mL at pH 5.0
4 1 0 3 mL at pH 6.0
5 1 0 3 mL at pH 7.0
1’ 0 1 3 mL at pH 3.0
2’ 0 1 3 mL at pH 4.0
3’ 0 1 3 mL at pH 5.0
4’ 0 1 3 mL at pH 6.0

5’ 0 1 3 mL at pH 7.0

In an additional test tube, 12 mL of the 0.1 M sucrose in water substrate solution were added and
the tube was grouped with the other 10 tubes in a hot water bath at about 37°C. The tubes were
allowed to heat to the temperature of the bath and the same reaction procedure as the previous
test was administered. Following the 10-minute reaction, the tubes were reheated for 5 minutes,
allowed to cool, and their absorbances values were taken at a wavelength of 540 nm. Tube 1 was
read against 1’ (which served as the blank), tube 2 was read against 2’ (which served as the
blank), and so on for all samples to acknowledge any hydrolysis caused by the acidity of the
medium. Data Table 2 (see Results section) was completed using the standard curve and a plot
was constructed with velocity on the y-axis and pH on the x-axis. The shape of the curve was
analyzed and the optimum pH was observed.

The next test was performed to ultimately find the KM and Vmax values of the enzymatic
reaction. Six test tubes were prepared in accordance with the lab protocol provided by Dr. Huang:

Tube Number mL of 0.1 M Sucrose in mL Acetate Buffer

Acetate Buffer
0 0.0 4.0
1 0.5 3.5
2 1.0 3.0
3 1.5 2.5
4 2.0 2.0
5 2.5 1.5

In an additional test tube, 8 mL of the β-fructofuranosidase in acetate buffer solution were added
and the tube was grouped with the other 6 tubes in a hot water bath at about 37°C. The tubes
were allowed to heat to the same temperature as the bath and the same reaction procedure as the
previous two tests was administered. Following the 10-minute reaction, the tubes were reheated
for 5 minutes, allowed to cool, and their absorbances values for tubes 1 through 5 were taken at a
wavelength of 540 nm. The values were measured against tube 0, which was set for zero
absorbance as the blank. The previously prepared standard curve was utilized in the completion
of Data Table 3 (see Results section). The initial data set involving tubes 1 through five was
plotted in terms of V versus [S], 1/V versus 1/[S], and V versus V/[S] and lines of best fit were
added in order to determine the best method in the evaluation of KM and Vmax.
V. Results

Part A: Absorbance vs. μmoles of Sucrose Equivalent to the Standard

Tube Number 1 2 3 4 5
Absorbance 0.081 0.100 0.210 0.270 0.450

Graph (1)

Notes: 1 mL of standard = 1 μmole of sucrose, and the equation derived from the standard curve,
y = 0.1673x – 0.0239, was used in formulating the following data tables and plots.

Part B: Influence of Enzyme Concentration on Enzymatic Reaction Velocity

Data Table 1:

Tube mL of Enzyme Used Sucrose Velocity (μmoles of

Number Absorbance (β-fructofuranosidase) Hydrolyzed in 10 sucrose hydrolyzed
minutes (μmoles) per minute)
mL of Enzyme Used (β-
Tube No. Absorbance fructofuranosidase)
1 0.050 0.5 0.4 0.04
2 0.102 1.0 0.8 0.08
3 0.172 1.5 1.2 0.12
4 0.221 2.0 1.5 0.15
5 0.307 2.5 2.0 0.20

Graph(2)

Notes: The standard curve equation previously found (y = 0.1673x – 0.0239) was used to find
the amount of sucrose hydrolyzed in 10 minutes (x) by using the corresponding absorbance
reading as the y-value. The plot data and the linear trend show that the enzymatic reaction is
first-order in respect to the enzyme concentration, which was only at constant, non-limiting
values.

Part C: Optimum pH Value of an Enzymatic Reaction

Data Table 2:

Absorbanc Sucrose Hydrolyzed

Tube e pH in Velocity (μmoles of
Number 10 minutes (μmoles) sucrose hydrolyzed per
minute)
1 0.002 3.0 0.155 0.0155
2 0.082 4.0 0.634 0.0634
3 0.152 5.0 1.053 0.1053
4 0.130 6.0 0.922 0.0922
5 0.004 7.0 0.167 0.0167

Notes: The standard curve equation previously found (y = 0.1673x – 0.0239) was used to find
the amount of sucrose hydrolyzed in 10 minutes (x) by using the corresponding absorbance
reading as the y-value. The graph showed the expected bell curve and the peak at pH 5.0
indicates the optimum pH.

Part D: Derivation of the KM and Vmax Values for β-fructofuranosidase

Data Table 3:

Sucrose Velocity (μmoles Molar

Tube Hydrolyzed of sucrose Concentration of
Absorbanc in 10
Number e minutes hydrolyzed per 1/v Substrate 1/[S]
(μmoles) minute, v)

calculatio
n is

1 0.003 0.161 0.0161 62.1 7.14 0.140

2 0.008 0.191 0.0191 52.4 14.3 0.070
3 0.018 0.250 0.0250 40.0 21.4 0.046
4 0.028 0.310 0.0310 32.3 28.6 0.035
5 0.029 0.316 0.0316 31.6 35.7 0.028

Notes: The standard curve equation previously found (y = 0.1673x – 0.0239) was used to find
the amount of sucrose hydrolyzed in 10 minutes (x) by using the corresponding absorbance
reading as the y-value. The Lineweaver-Burk plot produced the trend line that was the most
accurate and practical for assessing the KM and Vmax of β-fructofuranosidase.

Substrate concentration calculation: use C1V1 = C2V2 ; Sucrose is 0.1 M mL used during this
experiment is 5 mL

Tube 1 = (0.1 M x 0.5 mL)/(5 mL) = 0.01 M

Tube 2 = (0.1 M x 1.0 mL)/(5 mL) = 0.02 M

Graph
Graph

Calculations:
y-intercept = 1/Vmax = 26.071
(1/Vmax)-1 = (26.071)-1 = 0.038 = Vmax
x-intercept = -1/KM = -0.094

-(-1/KM)-1 = -(-0.094)-1 = 10.6 = KM

Graph

Calculations:
y-intercept = Vmax = 0.003

slope = KM = -(-0.0655) = 0.0655 M.

VI. Discussion and Conclusion

Overall, the Kinetics of β-Fructofuranosidase experiment was successful. The primary goals, to
ascertain how specific environmental factors influence enzymatic reaction velocity through
analysis of the kinetics and to determine the Michaelis constant (KM) and the maximum velocity
(Vmax) of the enzymatic system, were accomplished. In Part A, the absorbance readings resulted
in a linear trend that produced a suitable standard equation for comparisons with the other tests.
In Part B, the change in enzyme concentration yielded a linear trend that indicated that the
reaction was first order with respect to enzyme concentration, which matched the prediction. In
Part C, the experimental optimum pH was 4.0, which was lower than the known optimum pH of
6.0. This could be due contamination of the tubes, inaccurate measurements, orfaulty preparation
of the buffers used. However, the graph still showed a bell-shaped curve,which adhered to the
expectation. In Part D, the general trends for each graph resembled thepredictions, but the data
appeared to deviate from the expectation. The KM and Vmax values didnot match the values
from the literature, which could have been caused by inaccurate measurements, contamination,
timing, or calculations.

Questions:

Answer (1)

The method is used to determine the sucrose hydrolysis involved creating a standard curve
relating the absorbance to the µmoles of sucrose that had been hydrolysed. However, different
concentration of sucrose were added to test tubes with different concentration of water. DNS
reagent also was added to the test tubes as it were boiling, and the absorbance was taken after
cooling the tubes. DNS reagent is used to determine the reducing sugar in a reaction, and because
the sucrose is non- reducing sugar, it must be hydrolyzed to glucose and fructose.
Dinitrosalicylic cloeimetric method is the another method that is used which is just a
modification to the DNS method.

Answer (2)

The data for my experiment showed that the Km was 2.8461 mM, the optimum PH was 4.0, and
Vmax was 0.4247µmoles/min.

Answer (3)

The velocity of the reaction falls near PH 7 when the catalyzed by β-fructofuranosidasecan be
explained by the involvement of the ionizable group in the enzyme. The imidazolium group, the
side chain of histidine, has a Pka around 7.4. when the PH of solution reach 7, the protonated
imidazolium group is deprotonated and causing the loss of the a proton from the functional group.
The histidine amino acid is a part of the enzyme active site and the loss of the proton means the
creation of a salt bridge.