Virtual Lab Manual

Enzyme Kinetics
Synopsis
In the Enzyme Kinetics Lab, you will learn how substrates are converted into products by
catalysis. You will also learn all about the kinetics of enzyme involving the
Michaelis-Menten equation and various rate constants, as well as DNA mutation and
hyperactivity. You will get to run experiments using the enzyme Alcohol Dehydrogenase on a
wild and mutant type to learn about Alcohol Flush Syndrome.

Use a spectrophotometer to measure enzyme reaction

In the Enzyme Kinetics lab, you will access a fully equipped workbench where you can
prepare the Alcohol Dehydrogenase reaction and measure the product of Acetylaldehyde
using a spectrophotometer. You will learn about the concept of spectrophotometry, how to
prepare a master mix and how to calculate dilution. You will try to prepare a reaction in a 1
ml cuvette and measure the amount of product formed using the spectrophotometer.

See it all on a molecular level

Supplementary 3D animations illustrate what happens at the molecular level when the
substrate and co-factor enter the active site. During the 3D animations, you will also
answer quiz questions to test your understanding of the concepts. The animations are
interactive, so you can identify the substrate by clicking on the different molecules.

Experiment freely and measure the results

For every measurement, you receive a progress curve displaying amounts of product formed
over time. You must then analyze the outcome data and plot your own Michaelis-Menten
graph to find the Km and Vmax for each enzyme. By comparing Km and Vmax values of the
wild type vs. mutant Alcohol Dehydrogenase, you will be able to understand the Alcohol
Flush Syndrome. With the newly added module of enzyme inhibition, you are asked to
perform different enzyme inhibition experiments using three different inhibitors. You can

1 Copyright Labster ApS 2020

All Rights Reserved
measure product formation using several inhibitor concentrations, extract the data, create
your own Lineweaver-Burk plot and solve the Ki.

Updated with a mathematically based simulator

We have recently upgraded the Enzyme Kinetics lab by implementing a mathematically
based simulator. This provides you with a larger flexibility in conducting the experiments,
allowing you to change parameters such as substrate concentrations, enzyme
concentrations, temperature or pH and receive the corresponding results. In this
semi-guided module, you can experiment with different parameters in order to find the
optimal temperature and pH to reach the highest initial reaction rate.

Upon completing the Enzyme Kinetics lab, you will be familiar with the kinetics of enzyme
Alcohol Dehydrogenase. Will you be able to use your newly acquired knowledge to perform
the experiment and analyze the data outcome? And can you apply your knowledge to the
real life example of Alcohol Dehydrogenase and the Alcohol Flush Syndrome?

Learning Objectives
At the end of this simulation, you will be able to…
● Understand the experimental design of enzyme kinetics
● Understand the Michaelis-Menten model of enzyme kinetics
● Analyze spectrophotometer data and calculate Km and Vmax
● Understand that kinetics of an enzyme can be modified by genetic mutations
● Understand inhibition kinetics by using several types of inhibitors

Techniques in Lab
● Spectrophotometry
● Data analysis of enzyme kinetics measurements

Theory
Enzyme
Enzymes are proteins that act as catalysts of specific reactions. By providing an alternative
reaction with a lower activation energy, they allow the reaction to proceed at a much higher
rate. It is important to note that enzymes do not change the equilibria of a reaction; they
can only increase the rate. Without the enzyme, the reaction would therefore still proceed
in the same direction; however, it would be slower, often a lot slower. Enzymes usually
increase reaction rates between 105 and 107 times. Enzymes are required in our body to
perform specific metabolic reactions. They are highly specific for their substrates, much
like a key is specific to a specific door. The specificity of enzymes for their substrates led
Emil Fischer to propose the so-called "lock and key" hypothesis in 1894. The "lock and key"

2 Copyright Labster ApS 2020

All Rights Reserved
hypothesis implies that enzymes are static molecules, which they are not. Another
mechanism called induced fit is the preferred model; according to this model, the enzyme
undergoes conformational changes when binding to the substrate, and these changes are
necessary for catalysis [1,2].

Figure 1: A reaction from a substrate to product is a transition from one energy state to
another. A transition state exists between the substrate and product. This state has a
higher energy level than both the substrate and product. A catalyst will lower this energy
level, so that the transition energy is reached more easily, and this results in a faster
reaction.

How does an enzyme lower the activation energy?

There are many different ways by which enzymes enhance the rate of their specific
reactions. When two substrates are transformed into one or more products, the enzyme
will bind both substrates, thereby ensuring that they are in close vicinity and correctly
oriented towards each other. When the reaction occurs without the enzyme, the 2
substrates have to hit each other from the correct angle and with enough energy (speed) to
overcome the much higher activation energy. When the substrates are bound by an enzyme,
these challenges are overcome by the structure of the enzyme. An important characteristic
of enzymes (and catalysts in general) is that they are not used up during the reaction. When
a reaction has been catalyzed by the enzyme, it is ready to convert another set of
substrates. [1]

3 Copyright Labster ApS 2020

All Rights Reserved
Enzyme kinetic assay
Enzyme kinetics is the study of enzyme mechanisms through determination of reaction
rates under varied conditions. The rate of a reaction is dependent on several factors
including the concentration of the substrate and the enzyme, temperature, pH and
presence of inhibitors.

Figure 2: Top: Michaelis-Menten saturation curve. Bottom: Progress curve of a general

enzymatic reaction.

Performing kinetic assays

The aim of a kinetic assay is to model the reaction rate, V, as a function of the substrate
concentration, [S], as illustrated in Figure 2. It is therefore necessary to measure the rate at
several different substrate concentrations. For each substrate concentration, a progress
curve showing the amount of product formed as a function of time is obtained. Such a
progress curve is illustrated in Figure 3. As the figure shows, the rate of the reaction is

4 Copyright Labster ApS 2020

All Rights Reserved
approximately constant early in the reaction; however, as the substrate is used up, the rate
decreases, and the progress curve reaches a plateau. Because [S] changes during the
reaction, it is common to measure the initial rates (V0) of the reactions and plot these
against the substrate concentration. V0 is measured as the slope of the progress curve in
the beginning of the reaction, when the progress curve is still linear [1].

The simplest model of V as a function of [S] is the Michaelis-Menten equation. Reactions

where the Michaelis-Menten equation can be applied show an increase in the reaction rate
when [S] is increased; however, this increase is diminished as the rate approaches
the maximum velocity, Vmax as seen in Figure 2. The Michaelis-Menten equation will be
further described on the following pages [1].

Factors affecting the reaction rate

Temperature and pH also affect the reaction rate. Enzymes have an optimum pH, which is
dependent on the composition of the enzyme. This is due to the properties of the amino
acid side chains of the enzyme: some side chains need to be protonated or deprotonated in
order to make a functional enzyme. Whether an amino acid side chain is protonated or
deprotonated depends on the pKa of the side chain and the pH of the solution. For
instance, histidine has a pKa of 6.0, which means that it will be mostly protonated at
pH<6.0 and mostly deprotonated at pH>6.0. Note that the pKa of side chains may be
altered by the environment; therefore the pKa of the side chains in enzymes is usually not
the same as of the free amino acids. Temperature also affects the reaction rate: an
increase in temperature leads to an increased reaction rate; however, at a certain
temperature, depending on the enzyme, the enzyme will start to denature; therefore, the
reaction rate will start decreasing above this temperature [1].

The simulator
In this case you will use a simulator to perform the kinetic experiments. This simulator is
based on a mathematical model, and it is therefore "perfect", i.e., it does not result in any
experimental errors. This is, of course, not how the outcome of an enzyme kinetic assay
would work in real life, where such perfect data would not be obtainable.

Michaelis-Menten
The Michaelis-Menten model is a simple model of an enzymatic reaction developed by
Leonor Michaelis and Maud Menten in 1913. The model is based on the following 2
assumptions:
● An enzymatic reaction proceeds in 2 steps: formation of an enzyme-substrate
complex, ES, and dissociation of the enzyme and the product.
● After a (very) short period of time, the concentration of the ES complex reaches a
steady state, where the rate of formation of ES equals the rate of its consumption.

The first assumption implies that the enzymatic reaction is made up of 4 different
reactions: formation of ES from E and S, dissociation of ES into E and S, dissociation of ES

5 Copyright Labster ApS 2020

All Rights Reserved
into E and P, and formation of ES from E and P. The rate of a reaction is usually measured
in the beginning of the reaction, where no significant amount of P has been formed;
therefore, the rate of formation of ES from E and P can be ignored. This results in the
following overall reaction (Figure 3.a).

Figure 3: Figure 3.a: Overall enzymatic reaction; Figure 3.b: Plot of initial rates of an
enzymatic reaction plotted against the substrate concentration, and a Michaelis-Menten
curve fitted to this plot. At low substrate concentrations, the curve is steep; however, at
higher concentrations, the curve reaches a plateau, and the rate approaches Vmax. The
interpretation of km is also clear from the figure; km is equal to the substrate
concentration where the reaction rate is ½ • Vmax. [1]

This reaction implies that the rate of formation of products, i.e., the reaction rate, is given
by V = k2 • [ES]. When almost all the enzyme is part of the enzyme-substrate complex, the
reaction approaches its maximum velocity (Vmax). In the above reaction, k2 is the
rate-limiting step, and Vmax can therefore be expressed as [E] • k2. The rate-limiting rate
constant is also called kcat, or the turnover number, and in the above reaction, kcat = k2.
This means, that Vmax = kcat • [E].

6 Copyright Labster ApS 2020

All Rights Reserved
Inhibitors
Enzyme inhibition
Enzyme inhibitors are molecules that decrease the activity of enzymes, and knowledge
about inhibitors can, for example, be used in developing drugs or in the study of
biochemical pathways, because inhibitors provide a way to interfere with these pathways.
Enzyme inhibitors can be either irreversible or reversible; irreversible inhibitors decrease
enzymatic activity by destroying the enzyme through various mechanisms, while reversible
inhibitors keep the enzyme functional. The inhibitors we will study here are reversible
inhibitors.

Types of inhibition
The mechanisms of enzyme inhibitors can be classified into 3 major groups: Competitive
inhibitors, uncompetitive inhibitors, and mixed inhibitors. Competitive inhibitors work by
binding to the active site of the enzyme in competition with the substrate; uncompetitive
inhibitors bind to the enzyme-substrate complex at a site distinct from the active site, but
they cannot bind to the enzyme alone, and mixed inhibitors can bind to both the enzyme and
the enzyme-substrate complex at a site distinct from the active site.

The mechanisms of enzyme inhibition can be thought of as an extension to the

Michaelis-Menten mechanism and competitive and un-competitive inhibition can be regarded
as a special case of mixed inhibition (see Figure 4.a), where KI and K’I are the dissociation
constants of the EI and ESI complex, respectively. Using the same approach as that used for
deriving the Michaelis-Menten equation), the following equation for mixed inhibition can be
obtained:

V0 = V max•[S] / Km•α+[S]•α’

where α = 1+[I]/KI and α’ = 1+[I]/K’I

Just like the Michealis-Menten equation, this equation can be rearranged to fit a
double-reciprocal plot:

1/V0 = α’/Vmax + Km•α/Vmax • 1/[S]

If α > 1 and α’ > 1, the inhibition is mixed; for competitive inhibition, α’ = 1; for uncompetitive
inhibition, α = 1. Thus, 3 different equations are obtained for the 3 different types of
inhibition, and a Lineweaver-Burk plot of the kinetic data can reveal the type of inhibition
that the inhibitor performs (see Figure 4.b, 4.c, and 4.d).

7 Copyright Labster ApS 2020

All Rights Reserved
Figure 4: Figure 4.a; The overall enzymatic reaction and the extension of the enzyme
inhibition mechanism. Figure 4b, c, and d; Lineweaver-Burk plots showing the 3 major types
of inhibition. If the y-intersect is the same, but the slopes differ, the inhibitor is competitive.
If both the slopes and the y-intersects differ, the inhibitor is mixed. If the slopes are the
same, but the y-intersects differ, the inhibitor is uncompetitive.

Methanol poisoning
The enzyme alcohol dehydrogenase is not completely specific for ethanol; it also catalyzes
the formation of aldehydes from other alcohols. One of these alcohols is methanol, which
is metabolized into formaldehyde and other toxic compounds that can cause blindness or
death. Methanol poisoning is quite common, and can be caused by the ingestion of
homemade alcohol. Methanol and ethanol are thus competitive substrates, and ethanol is
actually used to prevent poising after the ingestion of methanol, because it inhibits ADH in
catalyzing the oxidation of this compound.

Master mix
For an experiment with 7 sample tubes and 5 reagents (4 of which are required at the same
concentration), the number of required pipette movements are shown. The figure below
shows the number of pipette movements necessary without the use of a master mix. For the
same experiment, a master mix is now used. Notice the drop in the number of pipette
movements - imagine even larger experiments and the time and workload difference
achieved.

8 Copyright Labster ApS 2020

All Rights Reserved
Figure 5: Two experimental setups with 7 sample tubes and 5 reagents (4 of which are
required at the same concentration). The number of required pipette movements is shown.
This figure shows the number of pipette movements necessary with and without the use of a
master mix.

When conducting an experiment with a large number of similar samples, it is common to

prepare a master mix to ease the workload in the lab. If all your samples have a similar
mixture of reagents with only 1 reagent varying from sample to sample, a master mix can be
prepared with all the common reagents (of course, this is only possible if all reagents are
required in the same concentrations). Now, when setting up your samples, you can pipette
from the master mix and then add your variable reagent, instead of mixing e.g., 4 different
reagents in correct amounts into each and every tube. The concept is illustrated in the figure
above.

In this enzyme kinetics case, at a specific substrate concentration, a master mix, containing
NAD+, ethanol, and buffer, can be prepared. When the enzyme is added, the reaction starts,
and the enzyme is therefore not included in the master mix. This is not a case where it is
crucial to use a master mix; however, the skill is essential for many applications, and it is
very useful in enzyme kinetics assays in general.

9 Copyright Labster ApS 2020

All Rights Reserved
Calculating substrate concentrations
In the experiment, you will need to calculate how much substrate to add to a tube to
obtain the desired substrate concentration. This can be done using the following formula:

C1 · V 1 = C2 · V 2

where C = concentration and V = volume. Remember to use the same units on both sides
of the equality sign!

Example: We have a stock solution of the substrate at 1 M, and we need a final

concentration of 200 mM in a volume of 500 μl; what is the volume of stock solution that
we need to add for achieving the desired concentration?

If the stock concentration is C1, C2 will be the desired final concentration and V2 will be the
final volume. This makes V1 the unknown element. We can now isolate V1, insert the known
values and calculate the volume:

Isolate: V 1 = (C2 · V 2)/C1

Insert and calculate: V 1 = (0.2 M · 0.0005l) / 1 M = 0.0001 L = 100 μl

Spectrophotometer
A spectrometer is an instrument that provides information about the intensity of radiated
energy. A spectrophotometer is an instrument that determines the ratio between the
intensity of light emitted from an internal source and that which passes through a given
solution. This ratio can be used to determine the concentration of dissolved molecules in a
sample.

Before a sample is measured, the spectrophotometer is set to measure at a certain

wavelength. The detected wavelength can be tuned to a value optimal for measuring a
specific compound.

On measurement, the spectrophotometer returns data as absorbance values (A).

Absorbance is calculated as log(I0/It), where I0 is the intensity of the incident light (light
falling on to a sample) and It is the intensity of the light that passes through the solution
and onto a photovoltaic detector.

The relationship between absorbance and concentration is linear and is described by

Beer-Lambert Law:
A = εcl

10 Copyright Labster ApS 2020

All Rights Reserved
where c is the concentration of the solution, l is the pathlength, otherwise described as the
distance traveled by light when passing through a solution (typically the width of a cuvette),
and ε is the extinction coefficient, which is specific for a compound.

Figure 5: Components of a spectrophotometer: Light emitted from the source passes

through the slit, letting only 1 specific wavelength through. This light partially passes
through the sample placed in a cuvette and the detector measures the intensity on the
other side of the cuvette.

In an example, we will measure concentrations of NADH. NADH has an absorption

maximum at 340 nm; therefore, the spectrophotometer is set to measure at this
wavelength. The extinction coefficient for NADH, ε = 6220 M-1cm-1. The pathlength is the
width of the cuvette, which is 1 cm.

There are certain limitations of the Beer-Lambert Law that spectrophotometer users must
consider. Some of these are related to technical issues; however, the law does have a real
limitation; because it only applies to dilute solutions. When the concentration of an
absorbing species increases, so does the frequency of physicochemical interactions among
the molecules. Thus, at a given concentration, the molecules will begin to affect the charge
distribution of the neighbor molecules. When this occurs, the relationship between
absorbance and concentration is no longer linear. As a rule of thumb, one should stay
below an absorption value of 1 when doing measurements.

Absorbance is inversely proportional to transmittance (light passing through a sample that

is not absorbed). When absorbance = 1, 10% of the light is transmitted through the sample,
at 2, 1% of the light is transmitted, and so on in a logarithmic trend.

11 Copyright Labster ApS 2020

All Rights Reserved