Enzyme Kinetics: Effect of Substrate Concentration on Enzyme

Activity

I. INTRODUCTION

K️inetics, in chemistry, is a branch of physical chemistry that involves an experimental study

concerned with understanding how fast or slow chemical reactions occur (Laidler, 2020). In other
words, it is the study of reaction rates and how they are affected. According to research, the rate
of a reaction pertains to the measure of the change in concentration of the disappearance of
reactants or the change in concentration of the appearance of products per unit of time (Odufalu
et al., 2013). Moreover, factors ️including concentration, temperature, pressure, enzyme activity,
and many more can impact the rate of a reaction.

This paper explicitly focuses on enzyme kinetics—a study of the rates of chemical reactions that
are catalyzed by enzymes. To briefly explain, catalysts are substances that speed up a chemical
reaction without undergoing any chemical change, while enzymes act as catalysts and proteins.
Furthermore, factors influencing the rates of enzyme-catalyzed reactions are described
mathematically using the following equations: (1) Michaelis-Menten and (2) Lineweaver-Burk
equation. The said equations are used to generally explain the velocity and gross mechanism of
enzyme-catalyzed reactions, and to determine important terms in enzyme kinetics (e.g., such as
Km and Vmax) through plots, respectively (Augustyn, 2019; Harvey, 2015). The components of
the Michaelis-Menten and Lineweaver-Burk equation are outlined and described in the
methodology.

Additionally, in enzyme kinetics, the reaction rate is measured in the experiment, and the effects
of varying reaction conditions are investigated. In essence, this provides insights into the catalytic
mechanism of an enzyme, its role in metabolism, how its activity is controlled in the cell, and how
drugs and poisons can inhibit its activity (Damji, 2019).

Therefore, this laboratory report aims to provide a deeper examination of the concept of enzyme
kinetics by conducting two parts of the experiment discussed below. In line with this, the objective
of this paper is to observe and understand the substrate concentration's effect on enzyme activity.

II. METHODOLOGY

The experiment was divided into two parts: (1) preparation of the standard curve and (2) enzyme
assay—executed in the same order.

Experiment 1: Preparation of Standard Curve

To begin the procedure, the following samples were concocted using varying amounts of glucose
solution, distilled water, and 1 mL dinitroalicylic acid (DNS) reagent, placed in large test tubes.
Refer to Table 1.

Table 1. Preparation of Standard Curve Samples

Test Tube No. Glucose Solution Distilled H2O, mL DNS, mL

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 (1 mg/mL), mL

Blank 0.00 5.00 1.00

1 0.10 4.90 1.00

2 0.50 4.50 1.00

3 1.00 4.00 1.00

4 2.00 2.00 1.00

5 3.00 3.00 1.00

6 4.00 1.00 1.00

7 5.00 0.00 1.00

These were mixed thoroughly, enclosed with aluminum foil, and put in a boiling water bath. After
five minutes, the tubes were taken out and submerged in lukewarm water. A 25-mL volumetric
flask was used to transfer the cooled samples; it was later diluted to its mark using distilled water
and homogenized. The next step involved the identification of the solutions' absorbance against a
blank at 540 nm with a Vis Spectrophotometer. Data processing followed this—a linear graph
involving absorbance and concentration of glucose was made (𝐴 = 𝑚𝑥 + 𝑏), where A is the
absorbance, m is the slope, x is the glucose concentration, and b is the y-intercept.
Consequently, the slope and y-intercept values were solved for, which are necessary for the
second part of the experiment.

Experiment 2: Enzyme Assay

The latter half of the experiment begins with a similar step to the former's. Seven samples were
prepared employing varying amounts of 5% pancreatic solution, 1 mg/ mL starch solution, 0.10
M Phosphate buffer, 0.9% NaCl solution, and .50 mL DNS; these were set in large test tubes.
Refer to Table 2.

Table 2. Enzyme Assay Samples

Test Tube No. 1 mg/mL, 5% Pancreatic 0.10 M NaCl, mL
Starch Solution Solution phosphate
(Substrate, mL) (Enzyme, mL) buffer, pH = 7
(Buffer, mL)

Blank 0.00 0.50 6.00 0.50

1 0.50 0.50 5.50 0.50

2 1.00 0.50 5.00 0.50

3 2.00 0.50 4.00 0.50

4 3.00 0.50 3.00 0.50

5 4.00 0.50 2.00 0.50

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 6 5.00 0.50 1.00 0.50

The water bath was prepared to be at 37°C, and the tubes were submerged for about 15 minutes.
After this, 1 mL of DNS was immediately added to each tube and enclosed with foil. Boiling water
bath followed this. After five minutes, the tubes were extracted and submerged under tap water to
cool. All the solutions are then transferred to a 25 mL volumetric flask, diluted to mark using
distilled water, and mixed generously. The absorbance of the solutions were yet again determined
against a blank at 540 nm with a Vis Spectrophotometer. Data processing trailed after with the
calculation of glucose concentration—via the standard curve equation (𝐴 − 𝑏)/𝑚—and velocity (
𝑉 = 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 (𝑚𝑔/𝑚𝐿)/15 𝑚𝑖𝑛𝑢𝑡𝑒𝑠). Graph of velocity and substrate and 1/v and
1/S were generated. Lastly, 𝐾𝑚 and 𝑉𝑚𝑎𝑥 from both graphs were attained through
Michaelis-Menten equation (𝑉 = 𝑉𝑎/𝐾𝑚 + 𝑎) and Lineweaver-Burke equation (𝑠𝑙𝑜𝑝𝑒 = 𝐾𝑚/𝑉)
for the former and the latter, respectively.

III. RESULTS

Table 3. Preparation of Standard Curve

Test Glucose Distilled DNS Final Concentration Absorbance
Tube Solution H20 (mL) (mL) Volume of Glucose
1mg/mL (mg/mL)
(mL) (x-axis)

Blank 0.00 4.90 1.00 25.00 0.00 0.00

1 0.10 4.90 1.00 25.00 0.004 -0.013

3 1.00 4.00 1.00 25.00 0.04 0.015

4 2.00 2.00 1.00 25.00 0.08 0.051

5 3.00 3.00 1.00 25.00 0.12 0.087

6 4.00 1.00 1.00 25.00 0.16 0.120

A = mc + b Y = 0.031x – 0.0161

m 0.031

b -0.0161

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 Figure 1. Absorbance vs. Concentration of Glucose

Table 4. Enzyme Assay

Test Substrate Enzyme Buffer NaCl DNS Final [S] Absorbance mg/ml Velocity
Tube 1mg/mL (mL) (mL) (mL) (mL) Volume (mg/mL) Glucose mg/mL*min
(mL)

Blank 0.00 0.50 6.00 0.50 1.00 25.00 0.00 0.046 0.00 0.00

1 0.50 0.50 5.50 0.50 1.00 25.00 0.02 0.030 0.0006 0.00004

2 1.00 0.50 5.00 0.50 1.00 25.00 0.04 0.034 0.0014 0.00009

3 2.00 0.50 4.00 0.50 1.00 25.00 0.08 0.046 0.0037 0.00025

4 3.00 0.50 3.00 0.50 1.00 25.00 0.12 0.047 0.0056 0.00037

5 4.00 0.50 2.00 0.50 1.00 25.00 0.16 0.001 0.0002 0.00001

6 5.00 0.50 1.00 0.50 1.00 25.00 0.20 0.096 0.0192 0.00128

IV. DISCUSSION

Experiment 1: Preparation of Standard Curve

Standard curves are graphs of light absorbance versus solution concentration that can be used to
determine the solute concentration in unknown samples and is always required when calculating
enzyme activity.

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 As observed, Table 3 exhibits the glucose solution, distilled H2O, DNS, and final volume in mL,
concentration of glucose (x-axis), and absorbance (y-axis). As such, all of these were measured
and calculated in all the seven test tubes. Meanwhile, Figure 1 shows the correlation of
absorbance to the concentration of glucose. Based on Table 3, it can be seen that the
absorbance and concentration of glucose is influenced by the amount of distilled H2O and
glucose solution.

Experiment 2: Enzyme Assay

V. CONCLUSION AND RECOMMENDATIONS

[What should be improved with this activity?]

VI. REFERENCES

[1] Augustyn, A. (2019). Michaelis-Menten kinetics | Definition & Facts. In Encyclopædia

Britannica. https://www.britannica.com/science/Michaelis-Menten-hypothesis

[2] Damji, F. (2019, February 19). Enzyme kinetics. Wikipedia; Wikimedia Foundation.
https://en.wikipedia.org/wiki/Enzyme_kinetics

[3] Harvey, D. (2015, December 26). The Equations of Enzyme Kinetics. Chemistry LibreTexts.
https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_
Maps/Map%3A_Physical_Chemistry_for_the_Biosciences_(Chang)/10%3A_Enzyme_Kin
etics/10.02%3A_The_Equations_of_Enzyme_Kinetics#:~:text=The%20Lineweaver%E2%
80%93Burk%20plot%20was

[4] Laidler, K. (2020). Chemical kinetics. In Encyclopædia Britannica.

https://www.britannica.com/science/chemical-kinetics

[5] Odufalu, F.-D., Chacha, P., Mudda, G., & Iskandar, A. (2013, October 2). 2.5: Reaction Rate.
Chemistry LibreTexts.
https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_
Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Kinetics/02%3A_R
eaction_Rates/2.05%3A_Reaction_Rate#:~:text=Reaction%20Rate%20is%20the%20me
asure

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