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Enzyme Kinematics. Effect of Substrate Concentration on Rate of Enzymatic activity.

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Introduction
Enzymes are important in living organisms and carry out different reactions. Some
biological reactions cannot happen or proceed at a reasonable rate without enzymes. They
operate as catalysts regulating the speed at which various chemical reaction occurs. Since the
enzyme acts as a catalyst in enzymatic reactions, they remain unchanged at the end of the
process (Eed, 2012). There are numerous types of enzymes, and their function is determined by
the amino acids. Chemical reactions take place within the active site of the enzyme, where
molecules bind to the available enzyme and undergo a chemical modification. The reaction
between an enzyme and substrate leads to the formation of enzyme-substrate complexes.
Enzymes accelerate the rate of reaction by decreasing the activation energy needed by the
substrate to produce a product (Grahame et al., 2015). During the transition-state arrangement,
enzymes decrease the activation energy by creating weak bonds that later become strong bonds
in the desired product. The enzyme-substrate complex is formed due to the substrate binding to
the active site of the enzyme. The substrate will consequently break off, becoming a product,
whilst the enzyme is neither modified nor consumed.
Enzymatic activity is affected by several factors such as pH, temperature, enzyme and
substrate concentration, activators, and inhibitors. These factors mainly affect the rate of
enzymatic reaction in different ways. The increase in substrate concentration causes an increase
in the rate of reaction of a certain quantity of enzyme. One significant parameter affecting the
rate of catalyzed-enzymatic reaction is the substrate concentration (Vitolo, 2020). The increase in
reaction speed is due to an increase in the collision frequency between the substrate and enzyme.
Therefore, enzyme-substrate complexes form rapidly or quickly, speeding up the process.
Nevertheless, there is a constrain, since all of the enzyme active sites ultimately become filled by
the available substrate. When this happens, the rate of reaction decreases significantly and does
not vary much as the substrate concentration is increased. At this point, there is so much
substrate and no active sites as they are saturated with the substrate. The reaction seems to be
inactive and the reaction can only continue if the substrate attached to the enzymes is released. In
this case, the enzyme is the limiting factor.
During a typical enzyme-substrate reaction, there is a gradual increase in the initial
velocity V0 with an increase in substrate concentration. Ultimately, a point is reached where or
beyond which V0 is independent of on [S] (Eed, 2012). Plotting a graph of V0 against the
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concentration of substrate on the X-axis generates a graph indicating that the concentration of the
substrate is directly proportional to V0 but to a particular point where the relationship seizes to
exist and velocity remains constant. Michaelis-Menten modeled that the first reaction involves an
enzyme combining reversibly with the substrate forming an enzyme-substrate complex (ES) in a
moderately fast reversible process (Vitolo, 2020). In a succeeding step, the ES complex breaks
down forming a product P and a free enzyme
E+ S ⇌ ES ⟶ E+ P
The Michaelis-Menten equation gives
V max [S ]
ν=
K M +[S]
 Vmax is the maximum velocity attained by the system at maximum substrate
concentrations. KM is the Michaelis constant representing the substrate concentration at 50%
reaction velocity (Vmax). [S] represents the substrate concentration (Grahame et al., 2015).

Figure 1: Michaelis-Menten Plot

The Lineweaver and Burke plot is a mathematical alteration of the Michaelis-Menten by
plotting a double inverse of reaction rate and substrate concentration.
1 Km 1 1
= × +
V 0 Vmax S Vmax

Enzymes that obey the Michaelis-Menten relationship generate a straight-line graph Figure 2.
The reciprocal of (1/Vo) is the y-axis, whereas the reciprocal (1/[s]) is the x-axis. The slope of
Km 1 1
the line gives , while is the intercept on the axis
V max V max V 0
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Figure 2: The Lineweaver- Burke plot

The equation above has a direct relationship with the equation of a line y=mx+c and thus can
be used to determine Km, Vmax, and Kcat.
The lab practical involved the determination of the effect of substrate concentration on
enzymatic reaction using enzyme cellobiose and substrate p-nitrophenyl. Reaction progress will
be tested at different time increments, stopping the enzymatic reaction and testing the absorbance
at that specific increment. Data will be used to generate various plots, such as the progress plot,
Michaelis-Menton Plot, and Lineweaver-Burk plot. An enzyme progress curve shows a
relationship between the product (x-axis) form and time (x-axis). Each of the substrate
concentrations will have a progress curve that will all be plotted on one graph to see the
relationship between them. The initial velocity will be determined using the progress curve using
the linear portion.
Objectives
1. To determine the effect of substrate concentration on the rate of enzymatic reaction
2. To use Michaelis-Menton Plot, and Lineweaver-Burk plot to determine Vmax and Km.

Method
At the start of the experiment, a blank solution and four standard solutions containing 12.5, 25,
50, and 100 nmol were prepared from a stock solution of p-nitrophenol. The absorbance values
of the blank and standards were measured using an absorbance wavelength of 410nm and the
results were recorded as shown in Table 1. From then on, eight 15 mL conical tubes were labeled
with the starting substrate concentration as indicated in Table 3. Appropriate amounts of the
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substrate were added followed by the transfer of the correct amount of buffer solutions into each
conical tube. Cuvettes were obtained and labeled with the concentration and times in increments
of 1 min, 2 min, 4 min, and 8 min. 500µl of stop solution was added to each cuvette followed by
750 µl of the enzyme and the timer started. Once the timer hit 1 minute, a micropipette was used
to transfer 500 µl of the stop solution into one of the cuvettes containing the stop solution
causing the enzymatic reaction to stop. The same procedure was repeated for 2, 4, and 8 min, and
the subsequent experiment was 2-8. Measurements were recorded all along and different tables
containing different datasets. Using the absorbance data and concentration, a calibration curve or
standard curve was generated. Also, using the equation M 1 V 1= M 2 V 2, the substrate
concentration was computed. The reaction progress curve, Michaelis-Menton Plot, and
Lineweaver- Burk Plot can be made using the data. These graphs are important when computing
Km, Vmax, and Kcat.

Results
Table 1: Amount of p-Nitrophenolate and the absorbance value at 410nm used to generate the
standard curve.
Standard Amount of p-nitrophenol (nmol) Absorbance at 410 nm
S1 0 0
S2 12.5 0.207
S3 25 0.407
S4 50 0.751
S5 100 1.218
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Standard curve of p-nitrophenol

0.8
f(x) = 0.01496 x + 0.014
0.7 R² = 0.997671937142303

0.6

0.5
Absorbance

0.4

0.3

0.2

0.1

0
0 10 20 30 40 50 60
Concentration of p-nitrophenol (nmol)

Figure 3: p-Nitrophenolate standard curve

The S5 (100 nmol) concentration was dropped when measuring the standard solution of
p-nitrophenol. The reason for not including the measurement when plotting the curve is that The
Beer-Lambert law is not consistent at higher concentrations since the linearity of the Beer’s law
is restricted due to instrumental and chemical. Individual analyte particles are no longer
independent of one another at high concentrations. The resultant interaction involving the analyte
particles can alter the absorptivity of the analyte. The analyte molecules are very close causing
significant deviations in the absorptivity Furthermore, the absorptivity of an analyte is influenced
by the refractive index of the solution. Since the refractive index (RI) of a solution fluctuates
with the changes in analyte concentration, the values of absorptivity (a) and molar absorptivity
(ε) may change. The refractive index and Beer's law are relatively constant and linear
respectively as fairly low analyte concentrations.

Table 2: Calculations of actual substrate concentration (mM)

Actual substrate concentration (mM)
M1 V 1
M 2=
V2
Experiment 1 1.5× 1.5 Experiment 5 0.125 ×1.5
M 2= =1 M 2= =0.0833
2.25 2.25
Experiment 2 1.0× 1.5 Experiment 6 0.060 ×1.5
M 2= =0.667 M 2= =0.04
2.25 2.25
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Experiment 3 0.5 ×1.5 Experiment 7 0.030 ×1.5

M 2= =0.33 M 2= =0.02
2.25 2.25
Experiment 4 0.250 ×1.5 Experiment 8 0.015 ×1.5
M 2= =0.17 M 2= =0.01
2.25 2.25

Table 3: Substrate and buffer volumes used and actual substrate concentration
Experiment Starting substrate Actual substrate Substrate added Buffer added
concentration (mM) concentration (mM) (mL) (mL)
1 1.5 mM 1.0 mM 1.5 0
2 1.0 mM 0.667 mM 1.0 0.5
3 0.5 mM 0.333 mM 0.50 1.0
4 0.250 mM 0.167 mM 0.250 1.25
5 0.125 mM 0.0833 mM 0.120 1.38
6 0.060 mM 0.04 mM 0.060 1.44
7 0.030 mM 0.02 mM 0.030 1.47
8 0.015 mM 0.01 mM 0.015 1.485

Table 4: Absorption data collected at the time increments and the amounts (Concentration) of the
substrate at the time increments.
substrate Absorbance Amount Absorbance Amount Absorbance Amount Absorbance Amount
t=1 min t=2 min t=4 min t= 8 min
1.5 0.418 26.93 0.782 51.2 1.035 68.067 1.305 86.067
1 0.319 20.33 0.526 34.13 0.863 56.6 1.295 85.4
0.5 0.193 11.93 0.323 20.6 0.544 35.33 0.913 59.93
0.25 0.109 6.33 0.151 9.13 0.249 15.67 0.418 26.93
0.125 0.0061 -0.527 0.085 4.73 0.133 7.93 0.215 13.4
0.06 0.044 2 0.057 2.87 0.086 4.8 0.21 13.07
0.03 0.039 1.667 0.046 2.13 0.053 2.6 0.078 4.267
0.015 0.033 1.27 0.031 1.13 0.047 2.2 0.052 2.53
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Table 5: The amounts (concentration) of the substrate in time increments.

Time 1.5 mM 1 mM 0.5 mM 0.25 mM 0.125 mM 0.06 mM 0.03 mM 0.015 mM
(min)
0 0 0 0 0 0 0 0 0
1 26.9 20.3 11.9 6.33 -0.527 2 1.67 1.27
2 51.2 34.1 20.6 9.13 4.73 2.87 2.13 1.13
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To determine the initial rate of the reaction, one needs to identify the linear portion of the progress versus the time curve. The velocity
V0 is then calculated from the slope of the linear portion of the curve.
[SI] [S2]
P ro d u c t f o rm e d

1.5 mM
1 mM
100 90
90
80

Product formed
80
70 70
60
50 60
40 50
30
20 40
10 30
0
0 1 2 3 4 5 6 7 8 9 20
Time
10
0
0 1 2 3 4 5 6 7 8 9
Time

The S1 curve involved picking the three colinear points while the first two points were selected for the S2 curve as indicated above
and were selected.
[S3] [S4]

70 0.5 mM 0.25 mM
60 30

Product formed
P roduct form e d

50
25
40
30 20
20
15
10
0 10
0 1 2 3 4 5 6 7 8 9
Time 5

0
0 1 2 3 4 5 6 7 8 9
Time

A similar concept was applied for [S3] and [S4] as the first two points in each curve were colinear and the rest discarded.
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[S5] [S6]

0.125 mM 0.06 mM
16 14
14 12

Product formed
12 10
Product formed

10
8
8
6
6
4
4
2 2

0 0
0 1 2 3 4Time 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9
Time

For [S5], the first and third points were colinear and were selected to determine the gradient. The first two points in [S6] were picked
to determine the slope.
[S7] [S8]
0.015 mM
0.03 mM

Product form e d
3
4.5
Product formed

4 2.5
3.5 2
3
1.5
2.5
2 1
1.5 0.5
1
0
0.5 0 1 2 3 4 5 6 7 8 9
0 Time
0 1 2 3 4 5 6 7 8 9

Time

Both progress curves for [S7] and [S8] involved selecting the first two points as they were colinear and were then used to find the V0
of the two reactions.
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Figure 4: Reaction progress curves of the experiments. The graphs indicate concentration with
time increments and are fitted with a trend line.

60
Progress curve against time to determine V0
f(x) = 25.6 x + 0.444444444444432
R² = 0.999096592346331

50

f(x) = 17.0666666666666 x + 1.08888888888889

40 R² = 0.987935219010196
Product formed

30

f(x) = 10.3 x + 0.544444444444434

R² = 0.991687554527704
20

10 f(x) = 4.56666666666666 x + 0.588888888888889

R² = 0.952483253264767
f(x)
f(x) == 2.36666666666666
1.43333333333333 xx −+ 0.964444444444444
0 f(x)
R² = ==0.667468764951071
1.06666666666667 x x++0.188888888888888
0.200000000000002
f(x)
R² = 0.566666666666665
0.950479780671694 0.233333333333335
0 R² = 0.90459363957597
0.5
R² = 0.662844036697243 1 1.5 2 2.5

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Time
Series2 Linear (Series2) Series 2
Linear (Series 2 ) Series 3 Linear (Series 3)
Series 4 Linear (Series 4) Series 5
Linear (Series 5 ) Seris 6 Linear (Seris 6 )
Series 7 Linear (Series 7 ) Series 8
Linear (Series 8)

Figure 5: Reaction progress curves of the experiments. Eight- and four-minute timestamps have
been removed to get better linearity and the regression coefficient R2.
Table 6: Table of actual substrate concentration and VO used to create the Michaelis-Menton
Plot.
Starting substrate Actual substrate concentration Initial velocity V 0
concentration (Mm) (mM)
1.5 mM 1.0 mM 25.6
1.0 mM 0.6666667 mM 17.07
0.5 mM 0.3333333 mM 10.3
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0.250 mM 0.1666667 mM 4.57

0.125 mM 0.0833333 mM 2.37
0.060 mM 0.04 mM 1.43
0.030 mM 0.02 mM 1.07
0.015 mM 0.01 mM 0.567

Michaelis-Menten Plot
30

25

20
V0 (nmol/min)

15

10

0
0 0.2 0.4 0.6 0.8 1 1.2
[S] (mM)

Figure 6: Michaelis-Menten Plot

Table 7: Table of 1/[S] (1/Mm) and 1/Vo used to create the Lineweaver-Burke Plot
1/[S] (1/Mm) 1/Vo
1 0.0390625
1.5 0.058582308
3 0.097087379
6 0.218818381
12 0.421940928
25 0.699300699
50 0.934579439
100 1.76366843
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Lineweaver-Burke Plot
2
1.8
f(x) = 0.0169615058440525 x + 0.108272633930655
1.6 R² = 0.976086342218481
1/VO (mMol/min)

1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 20 40 60 80 100 120
1/[S] (1/mM)

Figure 7: Lineweaver-Burke Plot

Calculating the Kinetic using the slope from the Lineweaver-Burk Plot.
y=0.017 x +0.1083
1
0.1083= ∴ V max=9.23 nmol/min
V max
Km
=0.017 ∴ Km=9.23× 0.017=0.157
V max
Discussion:
The lab practical involved the determination of the effect of substrate concentration on
enzymatic activity. Results from Table 4 indicate that substrate and enzyme reactions are directly
proportional in that as the concentration of substrate increases there is a subsequent increase in
the enzymatic reaction. Figure 4 shows a graphical representation of what is taking place
indicating a proportionate trend between substrate concentration and enzyme activity leading to
increasing in the product. The results agree with the literature that the rate of an enzyme reaction
increases with an increase in the substrate concentration in the presence of a specified amount of
enzyme. The frequency at which the enzyme and the substrate collide intensifies as the
concentration of the enzyme increases. Consequently, the enzyme-substrate complexes develop
faster, and reaction rates rise. However, as the reaction proceeds, linearity is lost after some time
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and the graph plateaus suggesting a decrease in the rate of reaction. This indicates an optimum
state referred to as the enzyme’s ideal substrate concentration.
Nevertheless, there is a limit, since there will ultimately be excess enzyme molecules
than the substrate, and increasing the concentration of substrate will no longer result in a
substantial increase in the reaction rate. There is the availability of more substrate that is attached
to every enzyme active site and no adequate enzyme molecules exist to bind to the substrate
molecules. At this point, the substrate has saturated all the enzyme molecules. The surplus
substrate cannot participate in the reaction until the attached substrate gets released, or gets
released without reacting. The Michaelis–Menten equation approximates the enzymatic
dynamics assuming that the concentration of the enzyme stays unchanged. The enzymes form an
enzyme-substrate complex with the substrate, resulting in the product's synthesis.
The values of Vmax and Km were calculated experimentally using the Michaelis–Menten
ploy. The value of Vmax was taken at the top where the graph attained a steady-state or plateaus
by drawing a line to the Y-axis. From Figure 6, the Michaelis–Menten plot, the reaction did not
reach the steady-state and thus it became quite difficult to compute Vmax as well as Km. This
can be attributed to using short time. Going further, the Lineweaver-Burke plot can be used to
compute the enzyme dynamic. The plot is the inverse of substrate concentration and reaction
rate. In enzymes that obey the Michaelis-Menten relationship, the reciprocal V0 versus [S] yields
Km
a straight-line straight line as indicated in Figure 7. The slope of the plot gives while the
¿ V max
1
intercept on the 1/V0 axis gives . From the calculations, V max ∧Km were found to be
V max
4.302 nmol /min ¿ 0.0473 respectively. The benefit of using the Lineweaver-Burk plot is it
determines Vmax more precisely, from a simple graph of V0 versus [S].
Conclusion
The experiment involved examining the effect of increasing substrate concentration on
enzymatic activity. The objectives of the experiment were met since the increase in the
concentration of the substrate caused a subsequent increase in the rate of reaction. The rate
increased because there was the availability of more substrates molecules that allowed for more
collisions and binding at the enzyme active sites forming the enzyme-substrate complex.
Therefore, increasing substrate concentration catalyzes the reaction because the active sites
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successively bind and produce the end product. However, the reaction plateaus showing a
decrease in reaction rate because the active sites are progressively decreasing due to enzyme-
substrate binding. The addition of more substrate does not cause any effect on the reaction.
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References
Eed, J. (2012). Factors affecting enzyme activity. Essai, 10(1), 19.
Grahame, D. A. S., Bryksa, B. C., & Yada, R. Y. (2015). Factors affecting enzyme activity.
In Improving and tailoring enzymes for food quality and functionality (pp. 11-55). Woodhead
Publishing.
Vitolo, M. (2020). A brief review on enzyme activity. World Journal of Pharmaceutical
Research, 9(2), 60-76.