Department of Biomedical Sciences

Department of Biomedical Sciences
Manual of Enzyme Kinetic Practical
Steady State Kinetic Study of Enzyme Activity
1. Background
In these sessions, you will investigate the steady state kinetics of yeast alcohol dehydrogenase
(YAD). You will have lectures explaining the theory behind this work and should refer to your
notes on these to help you understand your study.
As its name suggests, YAD catalyses the dehydrogenation of alcohols. The reaction is shown
below. YAD can use several different alcohols as substrates. The R group in the reaction below
could be H-, CH3, etc (see Table 1).
R-CH2OH + NAD+ <----> R-CHO + NADH + H+
The NADH generated absorbs light at 340nm and we use this property as a signal for the
progress of the reaction. Using a spectrophotometer, we observe the increase in absorbance at
340nm (A340) associated with production of NADH.
2. Experimental Objectives
The aim of these experiments is to determine some of the properties of an enzyme (YAD) by
measuring its rate of catalysis under steady state conditions. In particular:
A. Determine the initial rate of YAD under the standard assay conditions i.e. with ethanol as
the substrate. For this value you can calculate the specific activity of YAD with ethanol
as a substrate (µmoles/min/mg protein)
B. Determine which of the alcohols provided are substrates for YAD and which are not (Non-
substrates) and explain the structural features required for a substrate of YAD and what
structural features may alter YAD activity with alcohols that are substrates.
C. Determine which alcohols are inhibitors of YAD oxidation of ethanol and suggest what
structural features make an alcohol an inhibitor of YAD.
D. Determine the kinetic properties of ethanol in the absence or presence of an inhibitor
(Km, Vmax, Kmapp, Ki).
3. Experimental Protocol
3.1. Determination of enzyme rate (refer to Appendix 1 for
preparation and calculation sheet)
3.1.1. Reagents and Apparatus
 Spectrophotometer
 plastic cuvettes (1cm light path)
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Department of Biomedical Sciences
Manual of Enzyme Kinetic Practical
 60 mM sodium pyrophosphate buffer pH8.5
 Ethanol and other test alcohols: 6M stock
 YAD: in 0.1% (w/v) bovine serum albumin (store on ice)
 30mM NAD (store on ice)
3.1.2. Protocol: Testing for substrates of YAD
1) Pipette the following reaction mixture components into a plastic cuvette:
a. 2.2ml H2O
b. 0.5ml pyrophosphate buffer
c. 0.1ml 30mM NAD
d. 0.1ml 6M ethanol
2) Mix the cell contents.
3) Ensure the spectrophotometer is on and set to 340nm.
4) Place the cuvette into the spectrophotometer.
5) Use this sample to set the spectrophotometer to zero or ‘measure blank’.
6) Start the reaction by adding and rapidly mixing 0.1ml of YAD solution.
7) Allow the spec to follow the reaction for long enough (60 seconds with 6 sec
interval) for you to calculate the initial rate. You should observe the absorbance
increasing as NADH is produced in the reaction.
8) Calculate the initial rate, V0 (expressed in Abs units per minute). Collate your
results in Table 1.
9) Carry out the assay in triplicate (i.e. repeat steps 1-7 twice) or until you obtain 3
results within ±10% of one another.
10) Convert V0 from Abs units/min to mol NADH/min. The extinction
coefficient () of NADH at 340nm is 6.22 mM-1cm-1.
11) Determine the initial rate of reaction for the other alcohols listed in Table
1. Following the protocol described in steps 1-9.
Table 1: List of alcohols to test as substrates for YAD
Alcohol Reaction 1 Reaction 2 Reaction 3 Average
(mol NADH/min)
Ethanol
CH3CH2OH
Methanol
CH3OH
Propan-1-ol
CH3CH2CH2OH
2-Chloroethanol
CH2ClCH2OH
1,2-Ethanediol
CH2OHCH2OH
Propan-2-ol
(Isopropanol)
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Department of Biomedical Sciences
Manual of Enzyme Kinetic Practical
CH3CHOHCH3
2-Propen-1-ol
(Allyl alcohol)
CH2=CHCH2OH
1-Cyclopropyl-
methanol
12) Calculate the specific activity of YAD for each alcohol. Specific activity is defined as
enzyme activity per mg of protein. In this case, the specific activity should be quoted in
mol/min/mg.
13) Use your results to divide the alcohols into 2 groups: substrates and non-substrates (fill
in the Table 2 below).
Table 2. Determinations of substrates and non-substrates of YAD
Alcohol Specific Enzyme
Activity/ µmol NADH
produced.min-1.mg
protein-1
Percentage of
activity with
ethanol as
substrate/ %
Substrate or Non-
substrate?
Ethanol
(200mM)
100% Substrate
Methanol
Propan-1-ol
Butan-1-ol
2-
Chloroethanol
1,2-Ethanediol
Propan-2-ol
(Isopropanol)
2-Propen-1-ol
(Allylalcohol)
1-Cyclopropyl-
methanol
3.1.3. Protocol: Testing for Inhibitors of YAD
1) Using the reaction mix recipe described in Section as the basis of your experiment, test
each of the alcohols that were not substrates (non-substrates) of YAD for their ability to
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Department of Biomedical Sciences
Manual of Enzyme Kinetic Practical
inhibit the reduction of ethanol by YAD. You will need to make the changes described in
points 2 and 3 (below).
2) Use a non-saturating concentration of ethanol (10mM).
a. Calculate how much you need to dilute the ethanol stock to achieve a final
concentration of 10mM, when 0.1ml of the diluted stock is added.
3) Measure the initial rate at the new concentration of ethanol in the absence of other
alcohols.
4) Measure the initial rate in the presence of each of the non-substrate alcohols by adding
0.1ml of the 6M stock of each of the alcohols you want to test to the reaction mix.
a. You will need to reduce the volume of H2O by 0.1ml to accommodate the extra
volume.
5) Keep a table of your results and use these results to determine which alcohols are
inhibitors of YAD (similar to Table 1).
6) Use your results to identify the inhibitor (fill in the Table 3 below).
Table 3. Identification of inhibitor of YAD using ethanol as the substrate
Alcohols Specific Enzyme
Activity/ µmol
NADH
produced.min-1.mg
protein-1
Percentage of
activity with
ethanol as
substrate/ %
Non-substrate or
inhibitor
Ethanol only
(10mM)
100% N/A
Ethanol with
Ethanol with
Ethanol with
3.2. Design a study to determine the kinetic properties of YAD
using ethanol as the substrate in the absence or presence of an
inhibitor (Km, Vmax, Kmapp, Ki).
1. Select ethanol and the inhibitor.
2. Using the methods of the type used in section , design a kinetic study to learn more
about the inhibitor.
3. For example, to determine the enzyme inhibition mode and kinetic parameters (Km,
Vmax, Kmapp, Ki) by using Lineweaver-burk plot.
4. Simulated raw data will be provided (See Appendix 2).
3.3. Lab Report
Your report should include the following data:
 A table showing the measured rate of NADH production for each alcohol at saturating
final concentration (200mM). The data should be presented with units of mol/min/mg
(i.e. mol NADH produced per minute per mg of YAD) and as a percentage of the activity
obtained with ethanol.
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