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RBM2560 Medical Biochemistry

Practical 3 – Enzyme Kinetics.

Practical 3

Enzyme kinetics: alcohol dehydrogenase (ADH)

Read through this practical carefully. Then, fill out the tables of volumes of substrate (ethanol)
(for substrate assay) to be added in the method. It would be good to prepare this prior to the
lab and have it checked by your peers and lab demonstrator before setting up the assays.
Please ensure that you are prepared for the practical class as the reactions you prepare will
be used by your classmates and precision and accuracy are important in these assays.

Introduction

Alcohol dehydrogenase is a zinc metalloenzyme of broad specificity. It can be obtained from a

wide range of cells and tissues such as yeast and horse liver. The enzyme has Zn2+ ions in its
catalytic site. The yeast enzyme is a tetramer of Mr 145,000. Each chain can bind one NAD+
and one Zn2+.

Binding of nucleotide cofactor (NAD+/NADH) causes conformational change in the enzyme

that enables ethanol (or acetaldehyde in the reverse reaction) to bind the enzyme.

The liver enzyme is a dimer with various isozymes.

The zinc ion is located at the bottom of a hydrophobic pocket. It is ligated to the main enzyme
by thiol sulfurs of cysteine groups on the dehydrogenase and also by a nitrogen.

The thiol groups are critical for the activity of the enzyme. The fourth ligand is a water
molecule, hydrogen bonded to the hydroxyl group of a serine. The nicotinamide ring of NAD+
is bound close to the zinc ion.
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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

When ethanol/acetaldehyde binds to the enzyme, it replaces water. The oxidation of alcohols
is an ordered mechanism, with the coenzyme binding first and the dissociation of the altered
coenzyme from the enzyme being the rate-limiting step.

REACTION

ADH

CH3CH2OH + NAD+ CH3CHO + NADH+ + H+

Ethanol Cofactor Acetaldehyde Reduced coenzyme

Objectives:

The objectives of this practical are:

• To measure the activity of yeast alcohol dehydrogenase (ADH) at various pH and substrate
concentrations.
• To estimate the optimum pH of yeast ADH.
• To determine the KM and Vmax of yeast ADH for ethanol.

Principle:

In this experiment the optimum pH and kinetic parameters will be worked out from the activity
of the enzyme at various pH and substrate concentrations.

The rate of formation of NADH during the initial stages of the reaction (initial velocity) will be
used as a measure of enzyme activity. NADH absorbs light at 340 nm, whereas NAD+ has
negligible absorbance at that wavelength.

ε340 = 6,220 M-1cm-1

Given the time required to perform the assay, the preparation of different aspects of the
assay and their addition to the 96-well microplate will be divided between groups:

(Group 1) standards and no enzyme control;

(Group 2) pH assay; and

(Groups 3/4) substrate concentration assay

It is VERY important that the different groups communicate and collaborate to ensure that
each assay is prepared accurately and dispensed into the correct wells on the microplate
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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

(see below). Good pipetting technique is critical, so if you are unsure speak to your
demonstrator or lab partners for support.

ALL of the results from each plate will be analysed by all students that contributed to that plate.
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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

Materials (per 8 students – 4 groups)

Technical staff: Please note NAD+ and alcohol dehydrogenase should be freshly prepared
just before the practical class and stored on ice.

1 mL NADH standard solution (0.5 mM)

500µL NAD+ (6 mM stock)

500µL 1 M C2H5OH = ethanol (1M in dH2O) (σ = 0.79 g/mL)

500µL of 0.1 M C2H5OH

2 mL buffer (0.1 M sodium phosphate, pH 8.0)

500µL buffer (0.1 M sodium phosphate, pH 4.5)

500µL buffer (0.1 M sodium phosphate, pH 5.0)

500µL buffer (0.1 M sodium phosphate, pH 6.0)

500µL buffer (0.1 M sodium phosphate, pH 10.0)

4 mL alcohol dehydrogenase (36.6μg /mL) (need to check with technical staff as to what
concentration is available on the day)

5 mL ddH2O

Equipment (per 8 students):

1 x 96 well microplate (reading at 340 nm)

VICTOR Nivo Multimode Microplate Reader (Perkin Elmer)

40 x 0.2 mL Eppendorf tubes

Tray for 0.2mL tubes

Adjustable pipettes (e.g. Gilson/Eppendorf, 200µL and 20µL)

Yellow pipette tips

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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

Method:

Ensure the microplate reader is pre-warmed to 37ºC

Group 1

PREPARATION OF NADH STANDARD SOLUTIONS

Standard curve

To determine how much NADH has been produced during the reactions you will need to
prepare a standard curve of Absorbance plotted against NADH concentrations.

The standard curve should cover the range 0.0 – 0.4 mM (final concentration) in 150 µL. You
should have six points for your graph – each done in TRIPLICATE. Pipette the required
amount of the standard NADH solution into each tube and make it up to 150 µL with water as
shown in the following table. Note that in the following table the final amount of NADH has
been calculated for you. Plot a graph of average Absorbance against µmoles NADH. What
title would you give this table?

µmoles NADH (final amount) 0.00 0.007 0.015 0.030 0.045 0.060
Volume of standard 0.5 mM NADH 0 15 30 60 90 120
solution (µL)
Volume of H2O (µL) 150 135 120 90 60 30

Absorbance1 (λ = 340 nm)

Absorbance2 (λ = 340 nm)
Absorbance3 (λ = 340 nm)

Average Absorbance (λ = 340 nm)

Controls

You will have to include controls in this enzyme kinetics practical class and in most other
experimental work you do in the future – so read this section carefully. Ask you demonstrator
or lecturer for advice if you do not understand what a control is and why you use controls in
experiments.
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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

What are experimental controls and why have them? Perhaps the best way to answer this
question is to look at examples of controls and how they are used. In today’s experiments you
you will be determining enzyme activity by measuring the amount of product (NADH) made as
a result of enzymic reduction of NAD+. However, to determine the amount of NADH (N)
produced by the enzyme during a reaction it is no use simply measuring the mount of NADH
present at the end of the reaction, as there will probably have been some NADH present at the
start! Thus the NADH present at the end of the reaction is made up of what was there at the
start and what has been produced by the action of the enzyme!

Therefore, the amount of NADH produced as a result of the reaction is equal to the amount of
NADH (Nt) present at the end of the reaction minus the amount that was present at the start
(N0). Nt - N0 is equal to the amount of NADH produced during the reaction. It is therefore
essential that you have zero-time controls to determine the amount of NADH actually
produced.

The zero-time controls are the absorbance of the reactions PRIOR to the addition of the
enzyme and commencement of the reaction. The initial absorbance of the plate, prior to the
assay will provide this value for you for all of your assay reactions.

What control would you need to test whether or not the NADH produced during the reaction
was the product of enzymatic breakdown of NADH and not some other reaction taking place
at the same time (eg: spontaneous reduction of NAD+)?

Hint: You will need a control that has no enzyme in it.

You will prepare a no-enzyme control as described in the table below.

How will you know that the absorbance change you measure for the reaction is due solely to
conversion of substrates? For example, there may be something else in the reaction (eg: a
contaminant) which produces an absorbance at 340 nm. What control(s) will be needed to test
for this?

Hint: You will need a control that has no substrates in it.

Which reaction in the assay will act as a no-substrate control?

You will also include an absorbance ‘BLANK’ to measure any absorbance of the plate + the
buffer.

This value should be subtracted from ALL values in your standards, controls and assay
prior to any subsequent analysis.
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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

Prepare the following Blank solution and No-Enzyme Control and add to the appropriate wells
on the 96-well microplate (see figure above).

CONTROL BLANK NEC

Sodium phosphate buffer (µL) pH 8.0 100 100

6 mM NAD+ (µL) 0 15

1 M Ethanol (µL) 0 15

Water (µL) 50 20

Enzyme stock (µL) Added by Instrument 0 0

Absorbance1 (λ = 340 nm)

Absorbance2 (λ = 340 nm)

Absorbance3 (λ = 340 nm)

Average Absorbance

IMPORTANT: Ensure that you subtract the average absorbance of the BLANK from ALL
of your other readings prior to subsequent analysis.

Group 2

pH Assay to determine optimum pH of ADH

In this experiment the pH of the buffer used in the reaction mix will be varied to assess the
effect of pH on the rate of catalysis by ADH.

Set up the following reaction mixes and add to the appropriate wells on the 96-well microplate
(refer to the figure above).
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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

pH 4.5 5.0 6.0 8.0 10.0

Sodium phosphate buffer (µL)

100 100 100 100 100
Check the pH

6 mM NAD+ (µL) 15 15 15 15 15

1 M Ethanol (µL) 15 15 15 15 15

Water (µL) 15 15 15 15 15

Enzyme stock (µL)

5 5 5 5 5
Added by Instrument

Absorbance1 (λ = 340 nm)

Absorbance2 (λ = 340 nm)

Absorbance3 (λ = 340 nm)

Average Absorbance

N µmoles (using standard curve)

Nt - N0 µmoles

Groups 3 & 4

Substrate concentration assay to work out the KM and Vmax of the enzyme

Choose a range of 9 substrate (ethanol) concentrations between 2 - 100 mM. As you will be
plotting these on Lineweaver-Burk (double reciprocal) plots you need to work out the inverse
of these values and check (with the aid of marking the X-axis on a plot) whether these points
are evenly spaced. If not, choose other substrate concentrations that will give you evenly
spaced points on this plot.

Calculate out volumes of ethanol to give final concentrations that you have selected in 150µL
(this includes 5 µL enzyme to be added later, so the total volume for each reaction in the table
should be 145 µL) for each reaction. Use the appropriate stock of ethanol so that the volume
of it added to the reaction mix is never less than 2 μL and never more than 30 μL. Record these
in the following table. CHECK THESE WITH YOUR CLASSMATES AND THE
DEMONSTRATOR BEFORE YOU PREPARE THEM.
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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

Dispense the reactions into the appropriate wells on the 96-well microplate (refer to figure
above).

Volumes required to make up 10 final substrate concentrations from 0 to 100 mM.

Ethanol 0 100
Concentration
(mM)
Sodium 100 100 100 100 100 100 100 100 100 100
phosphate
buffer (µL)
pH 8.0
6 mM NAD+ 15 15 15 15 15 15 15 15 15 15
(µL)
100 mM 0 0
Ethanol (µL)
1 M Ethanol 0 15
(µL)
Water (µL) 30 15

Enzyme stock 5 5 5 5 5 5 5 5 5 5
(µL)
Added by
Instrument
Absorbance1
(λ = 340 nm)
Absorbance2
(λ = 340 nm)
Absorbance3
(λ = 340 nm)
Average
Absorbance
N µmoles
(using
standard
curve)
Nt - N0
µmoles

What is the importance of the tube with no ethanol in it?

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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

Collection of Data
Once your plate has been prepared you will perform your assays on the VICTOR Nivo
Multimode Microplate Reader (Perkin Elmer). This instrument has been set up to perform your
assays at 37ºC (why this temperature?) and measure the absorbance of each reaction for 2
minutes after the addition of the ADH enzyme.

As each assay is conducted independently, this can take some time (15 reactions in triplicate
for 2 minutes each!). While your data is being collected, you are strongly advised to work on
the analysis of data (see below) using the ‘test data’ provided on VU Collaborate. The
calculations can seem a bit daunting the first time you do this type of assay, so the support of
your peers and your demonstrator can be valuable before you start to analyse your own data.

After adding your plate in the correct orientation in the reader, you will run the following preset
programs in the following order:

1. RBM2560 Enzyme Assay – Initial Read

This program will measure the absorbance at 340nm for all of the samples on the plate. The data from this
run will be used for the standard curve, the zero time controls for all assays and the absorbance of the blanks.

2. RBM 2560 Enzyme Assay – Assays

This program will add the ADH enzyme to the pH and [substrate] wells, shake the plate briefly and then
measure the absorbance at 340nm every 10 seconds for 12 readings.

3. RBM2560 Enzyme Assay – Final Read

This program will read the absorbance at 340nm of the no enzyme control.

The data will be supplied to you as 3 separate Excel files containing the measured absorbance
values and the time points for the assays. If you have any trouble accessing the data, please
contact your lab demonstrator or lecturer as soon as possible.
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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

Treatment of Data
Graphing and calculations advice
For each pH or substrate concentration, do the following to get the values of specific
enzyme activity (v0) for every reaction you carried out:

1. Generate a standard curve of NADH by plotting the A340 values (don’t forget to subtract
the blank) against the µmoles of NADH. Generate the equation of the line using Excel
or similar graphing software.
2. Use the equation of the standard curve and the absorbance values of your assays (after
subtracting the blank and zero time controls) to determine the amount of NADH produced
at each time point in the assays. DO NOT EXTRAPOLATE THE STANDARD CURVE.
If the measured absorbance is not on the standard curve, then you cannot use it.
3. For each pH or substrate concentration tested, plot the amount of NADH on the Y-axis
vs. time (sec) on the X-axis for the reaction.
4. Draw a curve of best fit, then draw a tangent to the initial, linear part of the curve.
5. Work out the rate at which NADH is produced in this initial part of the reaction in µmol
NADH/minute (initial velocity). This is the gradient of that line!
6. Knowing that the stoichiometric relationship between NADH and acetaldehyde is 1:1
(see reaction in the Introduction) this will be the same as µmoles acetaldehyde formed
per second – convert this to µmoles acetaldehyde formed per minute.
7. Using the value of the stock enzyme concentration and the volume used, work out the
number of µg of enzyme used in the assay. (C x V)
8. Convert this to mg of enzyme used.
9. If (Step 6) µmoles of acetaldehyde were formed per minute by (Step 8) mg of enzyme,
how many µmoles of acetaldehyde per minute would be formed by 1 mg of enzyme?
10. This is the value for specific enzyme activity (v0) in µmoles NADH/min/mg. Work this out
for all pH values and substrate concentrations. Record the values you obtained in the pH
and substrate concentration assay tables above.
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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

Having calculated all of the v0 values:

For the pH Assay

1. Plot the V0 for each different pH against the pH of the reaction

2. Ensure you provide an appropriate title and label each axis, including units (where needed).
3. Present this graph in the results section of your report

For the Substrate Concentration Assay

1. Take the reciprocal of each substrate concentration (1/[S]) and record that in the table
below.
2. Record each of the v0 values you calculated above.
3. Take the reciprocal of each v0 value (1/v0) and record it.
4. Plot the 1/[S] on the X-axis and 1/ v0 on the Y axis. Draw a line of best fit (trendline in MS
Excel) This is your Lineweaver-Burk plot.
5. Work out the Vmax and KM for ethanol binding to this enzyme (refer to lecture notes or
textbook on how to get these values using the graph).

Amount of acetaldehyde produced for each reaction and parameters calculated for
Lineweaver-Burk plot

Final conc. of 0 100

substrate
(C2H5OH) (mM)
→

Specific activity - v0
(µmoles
NADH/min/mg
enzyme)
1/[S]
(1/mM)
1/v0
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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

Laboratory report submission – THIS IS AN ASSESSED PRACTICAL REPORT

Please submit your report to the appropriate Dropbox on VU Collaborate by the due
date.

The report should comprise the following

1. Introduction & Aims (2)

2. Methods
3. Results (5)
4. Discussion (3)
5. Conclusion
6. References (if any).

See below for details.

CHECKLIST FOR PRACTICAL REPORT

INTRODUCTION AND AIMS

• What sort of enzyme is alcohol dehydrogenase?

• Explain (in words) the reaction it catalyses.
• Write the metal ion that is bound to it and what groups it is bound to in the enzyme.
• State whether the substrate or cofactor has to bind the enzyme first and why.
• State the aims of the experiment.

METHODS
• Simply refer to the lab manual unless there were any changes.

RESULTS

Standard Curve

• Table with all sets of A340 recordings.

• Plot of A340 vs. [NADH]
• Include a title for the plot, labels for axes with labels and units (where relevant).

Controls
• Table with A340 recordings for the blank and no enzyme controls
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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

pH Assay

• Table with all sets of A340 recordings and calculations to convert these into specific
activities.
• Graphs used to determine the initial velocity of each reaction condition.
• Plot of pH vs V0

[Substrate] Assay
• Table with all sets of A340 recordings and calculations to convert these into specific
activities.
• Graphs used to determine the initial velocity of each reaction condition.
• Lineweaver-Burk Plot of 1/[S] vs 1/V0
• The calculated Vmax and Km for ADH

DISCUSSION

• Explain what you did in the practical.

• Describe the type of results you were expecting for the initial velocity plots and how
you expected these to change with changes in pH or increasing substrate
concentrations.
• Discuss the importance of the controls in the experiment and what they told you about
the validity of your results.
• Discuss the reproducibility of your replicates and why it was important to include
these.
• If you did not get results as expected, suggest possible reasons why not and
suggestions for improvements in future experiments. Don’t use phrases like
‘experimental error’, describe what might have gone wrong.
• Explain why initial velocity measurements for enzyme assays are usually more
accurate than end-point determinations.
• Explain why it is not enough to just plot absorbance changes vs. substrate
concentrations i.e. why you had to do all those calculations!
• Compare any published kinetic values of yeast ADH with your results and
conclusions. Comment on the accuracy (or otherwise) of your experiments.
• In the human body, alcohol is mainly metabolized by ADH in liver cytoplasm and
through the microsomal ethanol oxidizing system (MEOS) of which the main enzyme
is CYP2E1. The KM of ADH for ethanol is 0.04 mM whilst that of CYP2E1 is 11 mM.
Which system is likely to be operating more effectively in a person caught drink-driving
with a blood alcohol concentration (BAC) of 20 mM? Explain your answer.
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RBM2560 Medical Biochemistry
Practical 3 – Enzyme Kinetics.

For interest only:

20 mM alcohol works out to be ~ .09 BAC. The legal limit in Australia is .05.

If you want to work it out yourself, here is some information: .09 BAC means .09% i.e.
g/dL. The m.w. of ethanol is 46 g/mol.

Conclusion

• Your conclusions from the experiment and summary of the results obtained
• What you have learnt from this practical experiment

References

• Cite any references used, using a standard format such as Harvard.