Table 1. Data obtained from the glucose standard curve.

Concentration (X) Absorbance (Y)

0 0

0.4 0.3

0.8 0.33

1.125 0.35

1.6 0.45

2 0.75

Y=0.2988X+ 0.062
[Abs]=0.2988[Glucose] + 0.062
[Glucose] = ([Abs]-0.062)/0.2988

1. Interpret the Graph.

The graph exhibits right-skewed distribution. As the temperature increases, the enzyme
activity also increases until it reaches its optimum temperature, which is 40oC. The increase in
enzyme activity may be attributed to the increase of kinetic energy of substrates which makes it
easy to form substrate enzyme complexes. The enzymes will start to denature beyond the
optimum temperature. Denaturation of enzyme causes conformational change of the enzyme’s
active site leading to the inability of the substrates to bind to it. This results in the impairment or
total loss of function of the enzyme.
 Table 2. Effect of temperature on Enzyme Activity.
Temperature Absorbance mg% glucose Enzyme Activity

0 0.030 0.107
0.00535
40 0.280 0.730
0.0365
60 0.021 0.137
0.00686

[Glucose] = ([Abs]-0.062)/0.2988
EA = Glucose conc. / 20 mins

Figure 1. Illustrated graph of the effect of temperature on enzyme activity.

2.Interpret the Graph

As the pH increases, the enzyme activity also increases up until the optimum pH, which
is 6.7. Within the optimum pH, the enzyme is most active due to the ionization of specific
functional groups in the active site, and the general formation of hydrogen bonds important for
the overall conformation of the enzyme. Beyond the optimum pH (very acidic or very basic),
there is decreased enzyme activity. In basic conditions, deprotonation of amino groups occur
and in acidic conditions, protonation of carboxylic acid groups occur.
- Deprotonation and protonation affects structural stability and enzyme
activity since it causes conformational changes in your enzyme.

Table 3. Effect of pH on Enzyme Activity.

pH Absorbance mg% glucose Enzyme Activity
 6.2 0.026 0.1205 0.00602
6.7 0.364 1.0107 0.05054
7.2 0.250 0.6292 0.03146
7.7 0.100 0.1272 0.00636
8.2 0.030 0.1071 0.00535

[Glucose] = ([Abs]-0.062)/0.2988
EA = Glucose conc. / 20 mins

Figure 2. Illustrated graph of the effect of pH on Enzyme Activity.

3. Explain the result.

The presence of chloride ions in the solution resulted in a higher absorbance value. This
means that enzyme activity increases in the presence of chloride ions.

Table 4. Amount of reducing substances produced in the presence and absence of

chloride ions.
 Chloride Absorbance mg% Enzyme
glucose activity
Absence 0.033 0.0971 0.00485
Presence 0.356 0.9839 0.04920

Reason: Amylase has an allosteric site for chloride ions. Since chloride is negatively charged, it
will bind to the positively charged lysine in the allosteric site. This causes a conformational
change to the enzyme, making it more active.

Chloride ion is a positive allosteric effector of salivary amylase. Allosteric effectors bind to
allosteric sites, and increases or decreases the activity of your enzyme. In the case of salivary
amylase, chloride ion increases its activity.

4. Influence of Substrate Concentration

Table 5. Influence of substrate concentration on Enzyme Activity.
TT [substrate] abs [glucose] Preformed Amount of true Velocity
glucose reducing glucose
mg/dl mg/dl equivalent mg/min
mg/dl
mg/dl

1 2 0.181 0.398 0.024 0.374

0.01496

2 4 0.293 0.773 0.048 0.725

0.029

3 5 0.430 1.232 0.06 1.172

0.04688

4 6 0.545 1.616 0.072 1.544

0.06176

5 8 0.590 1.767 0.096 1.671

0.06684

6 10 0.675 2.052 0.12 1.932

0.07728

7 Same amt 0.026 0.120 --- ---

as TT6

Note: Test tube 7 = same amount of substrate with 6 but the enzyme was denatured
(since it was boiled)
We first calculated for the amount of glucose for each test tube using the equation:
[Glucose] = ([Abs]-0.062)/0.2988

To compute for pre-formed glucose, we used the values obtained from TT6 and TT7. Both test
tubes have the same amount of substrate. In TT7, the enzyme was denatured since it was
boiled. Thus, no reaction happened. We can assume that the amount of glucose detected in
TT7 is just the pre-formed glucose since no reaction happened. Hence, we can say that the
amount of pre-formed glucose in TT7 is equal to the amount of pre-formed glucose in TT6 since
they have the same amount of substrate. We then computed for the pre-formed glucose in the
other test tubes by dividing pre-formed glucose of TT6, which is 0.12 by the total amount of
substrate, which is 10 then multiplied it by 100. We got 1.2% which is the fixed amount of
pre-formed glucose in all test tubes.

Pre-formed glucose is the amount of glucose that is present in your starch even before the
enzyme is added.

To get the TOTAL amount of pre-formed glucose in each test tube, we just multiplied the amount
of substrate by 1.2%

For the true reducing glucose equivalent, we just subtracted pre-formed glucose from the total
amount of glucose that was detected.

Velocity or the rate = true glucose/25mins

Estimate Km
 Km = 1/2Vmax
Lineweaver Burke

From the given data, the Vmax for the Michaelis-Menten plot cannot be determined. As
such, ½ Vmax as well as Km also cannot be determined as both values are derivatives of
Vmax. The inability to find the value of Vmax can be attributed to the lack of samples. As seen
in the Michaelis-Menten plot, the graph does not display the characteristic plateau, denoting that
the speed of the reaction can no longer be influenced by an increase in the substrate
concentration. An alternative to getting the Vmax or Km would be assuming that the final
velocity of the Michaelis-Menten plot is the Vmax, i.e. TT6: 0.07728 mg/min.

Study Guide Questions:

1. For the effect of temperature, relate the shape of the curve to the effect of
temperature on reaction rates in general and on the enzyme structure.

Based on the graph, the optimum temperature for salivary amylase is 40°C.
Enzymes typically function as catalysts within the normal body temperature (35 - 40°C).
Below the optimum temperature, enzyme activity increases as temperature increases.
This is because high temperatures supply additional kinetic energy to the system. At
optimum temperature, enzyme activity reaches its peak. That is, the maximum amount of
substrate is converted to product per unit time. Above the optimum temperature, enzyme
activity decreases due to heat denaturation of the enzyme.

2. For the effect of pH, determine the optimum pH for salivary amylase activity.
Relate the shape of the curve to the effect of pH on reaction rates in general and
on enzyme structure.
 Based on the results, the optimum pH for salivary amylase is 6.7. At pH values
below 6.7, but approaching the optimal pH, the enzyme activity of salivary amylase also
increases. This is because the amino groups in the active site of the enzyme are
deionizing below the optimal pH. Once the pH reaches 6.7, the enzyme activity of
amylase is at its highest. However, as the pH increases past 6.7, the enzyme activity of
amylase decreases because the amino terminal at the active site is being deprotonated
at this point. Therefore, the curve of the effect of pH on reaction rates will be
bell-shaped.

3. For the effect of Chloride ion, interpret your data in terms of the possible
interaction of Chloride with the enzyme.

Binding to allosteric sites either increases or decreases the affinity of the enzyme
to the substrate. At temperatures near and above the optimum for salivary amylase
activity, an increase in the concentration or the presence of sodium chloride increases
amylase activity. Chloride ion is a positive allosteric effector of Salivary Amylase. It can
bind to Amylase’s allosteric site, increasing the total enzyme activity.

4. For the effect of substrate concentration, which is the more accurate method for
estimating Km? Why? State the significance of the Km value obtained.

The Lineweaver-Burke plot is more accurate in estimating the Km, since it

produces a more linear graph than the Michaelis-Menten. Moreover, Km and Vmax
cannot be computed in the Michaelis-Menten, since the experiment only has a limited
amount of substrate. Km value needs to be obtained in order to determine the affinity of
the substrate to the enzyme.

The lower the Km value the higher the enzyme's affinity for the substrate and
vice versa. This is because a high Km means that it takes a LOT of substrate to achieve
half Vmax.