Enzyme Kinetics

BIOLOGY 3 Lab Report 1

20313756 Clarissa Ngai En Ying

20317536 Emily Cheah Xing Roong

20301747 Yasmin Lau Yee

20311605 Kim Yeonjun

20262604 Aini Ahmed Nashid

Lecturer in charge: Ms. Jane Davies

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 TABLE OF CONTENTS

1.0 Introduction 3
1.1 Background Theory 3
2.0 Objectives 6
3.0 Methodology 6
3.1 Procedure 6
4.0 Results 8
4.1 Optimal conditions of ADH Enzyme 8
4.2 Km and Vmax values of ADH 8
5.0 Discussion 12
5.1 Performing Enzyme Kinetic Assays 12
5.2 Km and Vmax values 14
5.2.1 Theories and Explanations 14
5.3 Role and Variants of Alcohol-metabolising enzymes 15
5.4 Treatments for Alcohol Flush Syndrome 16
5.5 Limitations and sources of error16
6.0 Conclusion 17
7.0 References 18

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1.0 Introduction
1.1 Background Theory

Enzymes are biological catalysts that are made of globular proteins. They provide an
alternative reaction route with lower activation energy as shown in Figure 1.0.
Enzymes lower the activation energy and increase the rate of reaction by bringing
substrates together in a correct orientation and angle towards each other, hence
decreasing the energy needed to overcome. Each enzyme has an active site that is
specific where the substrate binds to it, forming an enzyme-substrate complex with
temporary bonds between the substrate and R-groups of the enzyme's amino acids.
When an enzyme converts substrates into products, it is not used up and can be reused
for further catalysis. (Enzyme Kinetics: Energy Levels (Introduction to Enzymes),
n.d.)

(Andruk, 2005)

Figure 1.0

There are many factors that affect the activity of enzymes and therefore the rate of
reaction, including the pH level, temperature, and the concentration of substrate.
(‘Factors Affecting Enzyme Activity | A-Level Biology Revision Notes’, 2016)

Firstly, enzymes can be sensitive to the pH level of the environment. This is due to
the Hᐩ interacting with the R-group of the enzyme’s amino acids, which can alter the
bonds present and the 3-dimensional shape. Some amino acid chains need to be

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protonated or deprotonated to act as functional enzymes. Specific enzymes have their
own optimum pH level, where enzyme activity and rate of reaction is at its maximum.
Too high or too low pH can alter the enzyme completely, making it denature. If the
original pH is restored, the enzyme activity returns to its original rate.

When temperature increases, the rate of reaction increases too. This is because
molecules gain kinetic energy and move faster, increasing the chances of successful
collision between substrate and enzyme, which leads to more substrate binding into
the enzyme. However, this increasing trend stops after the optimum value. After this
point, denaturation of enzyme occurs because the tertiary/quaternary structure breaks
and the active site is distorted.

Rate of reaction increases as the substrate concentration increases. This is due to the
increased chances of favorable collision between the enzyme and substrate, forming
more enzyme-substrate complexes and then products. Like temperature, this
increasing trend continues until the point of saturation, where concentration of
enzyme becomes the limiting factor. After this point, the rate of reaction plateaus,
which means increasing substrate concentration further has no effect.

Not only do enzymes increase rate of reactions depending on these factors, but they
also play a crucial role in metabolic reactions in our body.

Upon alcohol intake, a healthy human body metabolises alcohol in the liver. Ethanol
is catalysed into toxic Acetaldehyde by the Alcohol dehydrogenase (ADH) enzyme
and an oxidising agent, [NAD+]. NAD+ (Nicotinamide Adenine Dinucleotide) is a
coenzyme that acts as an electron acceptor, thereby receiving 2 electrons and a H+
proton from ethanol. Acetaldehyde, formed as a result of this oxidation reaction, is
further broken down into Acetic acid, CH3COOH, using the Acetaldehyde
Dehydrogenase (ALDH) enzyme. (Kimura et al., 2015) Figure 2.0 illustrates the
chemical process of ethanol metabolism.

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 Figure 2.0 Metabolism of Ethanol

(Roth, 2005)

Alcohol Flush Syndrome (AFS), also known as the “Asian Glow” syndrome, is a
genetic disease which occurs due to DNA mutation, as well as increase in activity
of mutant ADH (i.e., ADHB1*2). Over 70% of the East Asian patients that
exhibit genetic polymorphism experience AFS. (University of California, San
Francisco, 2020) It is commonly characterised by severe flushing, nausea,
headaches, palpitations, or hangovers, even with very minimal alcohol
consumption. Medications and prescriptions such as oral antihistamines can be
given and are effective to some extent, in terms of easing the symptoms of AFS.
However, future targeted therapies and advancements relative to AFS are still yet
to be explored. (Matsumura et al., 2019).

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2.0 Objectives

The aim of this enzyme kinetic assay is to identify and distinguish the presence and activity
of different ADH (wild/mutant) enzymes, by analysing spectrophotometer data to find the
optimum pH and temperature for ADH, and by calculating K m and Vmax values based on
experimental designs i.e., Michaelis-Menten and Lineweaver-Burk model, in order to
detect Alcohol Flush Syndrome (AFS) in a patient.

3.0 Methodology
3.1 Procedure
Before conducting the experiment, it was ensured that lab coats and safety gloves
were worn as a safety precaution.

To prepare the master mix, a 15-mL test tube was collected from the tube box and
placed in the tube rack. Using a pipette, Ethanol, NAD+ and pH buffer were added to
the same test tube. Pipette tips were changed between the transfer of each sample to
avoid carry-over contamination. A reference blank cuvette that holds no analyte of
interest was prepared and placed in the spectrophotometer, setting an absorbance
baseline. A new cuvette was then obtained and placed into the rack, and the master
mix and ADH1B*1 enzyme were added using a pipette. It was ensured that the
master mix was added before the enzyme, then the cuvette was immediately inserted
into the spectrophotometer. By the NADH absorption chart seen on the monitor, the
optimal wavelength to measure NADH was then found.

Next, a couple of cuvettes were prepared, each added with 16µM ethanol and varying
concentrations of ADH (Alcohol Dehydrogenase) enzyme. Each cuvette was then put
into the spectrophotometer, and NADH absorption charts with different enzyme
concentrations were obtained. The optimal enzyme concentration to break down the
16µM ethanol in the first 130 seconds was determined using the charts and recorded
in Table 1.0. Similarly, the optimal pH and temperature of the ADH enzyme were
also determined by preparing multiple cuvettes with the same master mix and

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enzyme, but under different temperatures and pH conditions. The temperature of the
reaction was modified by incubating the cuvette with enzyme, ethanol, and buffer at a
particular temperature whilst measuring the reaction rate. To adjust the pH level, a
specific amount of acids or bases were poured into the solution in the cuvette.
Looking at the NADH absorption chart, optimal pH level and temperature for the
enzyme activity was set and recorded into Table 1.0.

Subsequently, wild type ADH enzyme and ADH enzyme extracted from a suspected
Alcohol Flush Syndrome (AFS) patient was prepared in the lab. The enzyme
concentration and pH level were kept constant at the optimal conditions of the ADH
enzyme that was determined beforehand. Then, a new cuvette was prepared and
placed in the cuvette rack. Using the pipette, the master mix containing ethanol,
NAD+ and buffer was added in, followed by the wild type ADH enzyme. The cuvette
was then placed in the spectrophotometer at room temperature (25℃) and the NADH
absorption chart was seen in the monitor. This process was then repeated with a total
of ten different initial substrate concentrations.

Using the values from NADH absorption chart, the plots needed for the Michaelis-
Menten graph and Lineweaver-Burk plot was calculated and presented in Table 2.0,
and the graphs were drawn accordingly. The value of Vmax and Km was also calculated
and recorded in Table 3.0. Another set of ten cuvettes was prepared, each with ADH
enzyme from the patient and ten different concentrations of substrate. They were
tested under the spectrophotometer in the same way cuvettes with wild ADH enzyme
was tested. The calculated plots for drawing Michaelis-Menten graph and
Lineweaver-Burk plot were recorded in Table 2.0. Vmax and Km values for mutant
ADH obtained was recorded in Table 3.0. Finally, comparisons were made between
the values of the two enzymes.

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4.0Results
4.1 Optimal conditions of ADH Enzyme

Table 1.0 Optimal conditions of Alcohol Dehydrogenase enzyme

Concentration (mg/mL) 0.1

pH 8.5

Temperature (°C) 44

4.2 Km and Vmax values of ADH

Table 2.0 Wild Type and Mutant ADH

Wild Type ADH Mutant Type ADH

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 Michaelis-Menten and Lineweaver-Burk graphs were plotted based on the results tables
illustrated above, and Vmax and Km values were calculated for both the Wild type ADH and
Mutant ADH.

Wild-Type ADH Calculations

Y= 0.888x+ 55.6

Figure 1. Lineweaver-Burk graph for wild type ADH illustrated above helps calculate
the exact Vmax value.

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Vmax= ¿ 17.98 µM /min ≈ 18 µM /min
0.0556

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Figure 2. Michaelis-Menten graph for Wild-type ADH showing the Vmax value, and Km
value that corresponds to ½ the Vmax.

Km = slope × Vmax

= 0.888 × 18

= 16µM

= 0.016 mM

Mutant-Type ADH Calculations

Y= 3.96x+ 2.08

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 Figure
3.

Lineweaver-Burk graph for mutant ADH illustrated above helps calculate the exact Vmax
value.

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V max =
0.00208

¿ 480.7 µM /min ≈ 480 µM /min

Figure 4. Michaelis-Menten graph of mutant ADH showing the Vmax value, and Km value
that corresponds to ½ the Vmax.

Km = slope × Vmax

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 = 3.96 × 480

= 1900.8 µM

= 1.9 mM

Table 3.0 Vmax, Km of wild and mutant ADH

Wild ADH Mutant ADH

Vmax 1/0.0556 = 18 µM/min 1/0.00208 = 480 µM/min

Km 0.888*18 = 16µM 3.96*480 = 1900.8 µM

In the experiment, the V max value obtained for the mutant ADH was 480 µM/min while
the V max value obtained from the wild type ADH was 18 µM/min (table 3.0).

The Km value for mutant ADH is 1900.8 µM whereas for wild type ADH it is 16µM.
Again, the K m value we obtained for the mutant ADH is much higher compared to the
wild type ADH (Table 3.0).

The higher Vmax and Km value of the patients ADH enzyme shows a similar function to
the mutant ADH suggesting that the patient has hyperactive ADH.

5.0Discussion
5.1 Performing Enzyme Kinetic Assays

Two graphs were utilised in this experiment to find the values of K m and Vmax which
are the Michaelis-Menten saturation curve and then the Lineweaver-Burk plot.

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In the Michaelis – Menten saturation curve it is mandatory to test and measure the
rate at different substrate concentrations as the graph is modelled V (reaction rate)
against [S] (substrate concentration). Initially the reaction rate increases at a
constant rate (said to be linear) until the reaction declines as a result of substrate
concentration being exhausted, at this point maximum velocity is reached as
shown. Km is equal to the [S] where reaction rate would be half of Vmax
(1/2Vmax). V0 then can be found using Michaelis – Menten equation which is
given below. (Michaelis-Menten - Labster Theory, n.d.)

An alternate direct method using the Lineweaver-Burk equation (Km - Labster

Theory, n.d.) is illustrated above where it is stated that 1/Vmax would be the y
intercept, which can be found using the graph and then Km is found using the
gradient. This method was applied to calculate the Km and Vmax values for both
mutant and wild type ADH for this experiment.

y = c + (m × χ)

The patient’s enzyme sample behaves similar to that of mutant ADH. This means
that he has a condition known as Alcohol flush syndrome. The mutation of the
enzyme ADH inactive and therefore after his liver converts ethanol to
acetaldehyde which, in fact is more toxic, is not converted to acetic acid that is

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 nontoxic. The accumulation of acetaldehyde in the blood stream even though the
patient had a small amount of alcohol will result such as rapid heart rate, flushing
of skin and behavior similar of those who are heavily intoxicated.

5.2 Km and Vmax values

5.2.1 Theories and Explanations

As shown in table 1.0, an optimum temperature of 44℃ for the ADH

enzyme was obtained. Even though the ADH enzyme functions optimally
at this temperature, it also works well at body temperature of 37℃, and
room temperature at 25℃. Spectrophotometers in most cases do not have
temperature settings and thus the experiment was carried out at normal
room temperature of 25℃ instead of the optimum temperature of 44℃.

In this experiment, V max is the maximum initial rate at which the enzyme is
saturated and indicates how fast the enzyme is able to catalyse the reaction
of breakdown of alcohol. (Welcome to the Enzyme Kinetics Lab - Labster
Theory, n.d.) The mutant ADH gave a higher V max value meaning that more
ethanol is being converted per minute compared to the wild type ADH
(Figure1 & 3).

K m is the concentration of substrate required for the enzyme to be

saturated and for it to reach ½ of the V max . A lower K m value means that
only a low concentration of substrate is required for the enzyme to be
saturated and for it to reach ½ the V max value. Hence, for wild type ADH
with a lower Km value, this indicates that it has a high affinity for the
substrate, ethanol. As for the mutant ADH, a much higher Km value was
obtained meaning that a high concentration of substrate is required to
saturate the enzyme and for it to reach the ½ the V max value. Thus, it has a
low affinity for the substrate, ethanol (Figure 2 &4). (Welcome to the
Enzyme Kinetics Lab - Labster Theory, n.d.)

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 The patient’s enzyme was tested and compared to the wild type and
mutant ADH. It reacted similarly to the mutant ADH where high
concentrations of ethanol were being converted per minute while showing
low affinity to ethanol. This proves that the patient is sensitive to even
small amounts of alcohol and therefore has alcohol flush syndrome.

5.3 Role and Variants of Alcohol-metabolising enzymes

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) metabolise

alcohol by various oxidative mechanisms. Genetic polymorphisms are different
variants of the ADH protein that alter kinetic properties of ADH and ALDH
enzymes, causing changes in acetaldehyde turnover and alcohol activity in the
body. (Kimura et al., 2015)

The isozyme ADH1B mainly consists of ADH1B*1 and ADH1B*2 alleles, which
differ by one amino acid residue. Pharmacological and pathophysiological effects
of alcohol depend on the range of alcohol concentration and its metabolites, as
well as the duration of exposure to these chemical substances. (Jain et al., 2019)
Based on genetic case studies, it may also be mediated by acetaldehyde
accumulation and hyperactivity of ADH gene polymorphisms, which suggest the
increase in rate of acetaldehyde generation. (Crabb et al., 2004) Table 4.0
compares ADH allele frequencies among different ethnic groups that exhibit
ADH1B*1 and ADH1B*2 gene polymorphisms. (Higuchi et al., 2004)

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 Table 4.0 Allele frequencies of ADH1B*1 and ADH1B*2
(Higuchi et al., 2004)

According to In-vitro studies, beta encoded subunits of ADH proteins such as the
ADH1B*2 allele are shown to be much more hyperactive upon ethanol
oxidisation, relative to subunits encoded by the ADH1B*1 allele. (Yoshida, 1994)
A study was conducted where Jewish individuals were given doses of ethanol by
intravenous methods of infusion. As a result, subjects with the ADH1B*2 allele
showed higher alcohol elimination rates, which is linked to higher acetaldehyde
production and flushing upon ethanol consumption, which is the Alcohol Flush
Syndrome. (Goedde et al., 1992)

Moreover, the kinetic differences shown in Table 3.0 are due to chemical
properties of the ADH1B genes. The ADH1B*1 allele contains arginine residue,
which forms Hydrogen bonds with pyrophosphate group of NAD+ and the
ADH1B*2 allele contains histidine residue, which does not form Hydrogen
bonds, and therefore is weakly bound to NAD+ leading to an increase in Km value
in mutant ADH.

5.4 Treatments for Alcohol Flush Syndrome

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 A possible way to treat AFS is the use of inhibitors as it serves to decrease the
activity of the mutant enzyme. The mechanisms are divided into three main types
of inhibitors:

1. Competitive inhibitors: decrease the activity by binding to the active site

of the enzyme.
2. Uncompetitive inhibitors: decrease activity by binding to the enzyme
substrate complex at different site apart from the active site.
3. Mixed inhibitors: decreases activity by binding itself to both the enzyme
or at different site apart from the active site.
The inhibitors listed are also known as reversible inhibitors. There are also
irreversible inhibitors which decrease activity by mechanisms.

Some treatments that are currently offered for alcohol syndrome include
histamine-2 (H2) blockers (Red Face from Alcohol: Causes, Symptoms, and
More, n.d.) which helps to reduce redness of the face. Other blockers such as
Pepcid, Zantac and Tagamet may also be prescribed to the patient. However,
these medications may disguise symptoms that could indicate and identify serious
problems. There is no treatment that offer a complete solution to alcohol flush
syndrome yet. The patient must limit their consumption of alcohol as a preventive
measure.

5.5 Limitations and sources of error

The few sources of error that may have an effect on the observed results include:

 Environmental conditions and malfunctions of spectrophotometer: such as

temperature and sun light exposure may interfere with results taken up from the
spectrophotometer by heating it up. Subsequently, heating the sample. (Sources of
Error in Spectrophotometry Lab - Google Search, n.d.).Moreover, contaminants
in the air such as chemical vapors will also cause a gradual reduce in the accuracy
of measurements that the spectrophotometer provide. Voltage fluctuations can
also affect the spectrophotometer. This device should be handled with great care.

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 (What Are the Right Environmental Conditions for My Spectrophotometer? |
Datacolor, n.d.). Malfunctioning of the photometer can discourse the substrate
blank from being constant which may then alter and interfere the reactions of
enzyme (Bisswanger, 2014).
 Calibration of pipette: pipetting of small volumes of solutions can lead to higher
uncertainty of values than that of pipetting of larger volumes (Bisswanger, 2014).
This could be avoided by recalibration by a person who is familiar with the
equipment.
 Errors found within sample: inaccurate methods of collection of sample from the
patient can also affect the procedure and ultimate results. Environmental
conditions such as humidity (moisture content) can cause color of sample to
change and contribute to variations in data measurement. (Sources of Error in
Spectrophotometry Lab - Google Search, n.d.)
 It is as equally important to make sure to use instruments that are in good
condition. Using cuvettes that have stains or scratches cuvettes may give rise to
errors. It may seem minor but this affects the amount of light that penetrates
through thus the measurements would be affected. (Biology - Enzyme Reaction
Rates - University of Birmingham, n.d.)

Conclusion

In conclusion, a spectrophotometer was used to find the activities of wild and mutant
ADH enzymes. The optimum pH and temperature for both enzymes were the same,
which were 8.5 and 44°C respectively.

Besides, according to Michaelis-Menten graph and Lineweaver-Burk graph, the

calculated Vmax and Km value of mutant ADH is higher than wild ADH enzyme. The
Vmax (480 µM/min) and Km (1900.8 µM) of mutant ADH is similar to the patient’s
enzyme sample. This have proved that the patient has mutant ADH enzyme, causing the
patient sensitive to small amount of alcohol, thus having alcohol flush syndrome.

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