Yeast Alcohol Dehydrogenase Substrate Specificity

Published Date: 02 Nov 2017

Introduction

This lab was carried out to compare the Km of three different substrate of the enzyme Alcohol Dehydrogenase, which is found in the Saccaromyces cereviciae Yeast. Yeast Alcohol Dehydrogenase is an allosteric oxidoreductase enzyme, which has a group specificity for molecules with an –OH functional group. It weighs about 141000 Da (Dickinson. 1976). It oxidized alcohols using βNAD and βNADH as electron acceptors (Worthington Biochemical Corporation. 2013). It has four equal sub – units, each with an active site, two sulphydryl groups and a histidine residue (Dickenson & Dickinson, 1976). The enzyme works best at pH7 at room temperature (Leskovac. 1975). Single chain alcohols which have between two and ten atoms are able to be cleaved by the enzyme’s active site (Schöpp. 1976). Two of the small active sites of Yeast Alcohol Dehydrogenase are used for coenzymes which allow the better production of βNADH and aldehydes. Yeast Alcohol Dehydrogenase is an allosteric enzyme, so it should be able to work with the three alcohols. However, ethanol should have the lowest Km value because it has the best shape to fit to the enzyme. Theoretically ethanol should have a Km of around 2.1mM after it has been in the Mass Spectrophotometer. Methanol should have a Km of around 130mM and Propan-1-ol should have a Km of around 140mM. The enzyme will catalyse the reaction and the absorbance of NADH will be recorded.

Ethanol + βNAD Acetaldehyde + βNADH

Methanol + βNAD Formaldehyde + βNADH

Propanol + βNAD Propionaldehyde + βNADH

(Trivic, et al. 1998)

The UV absorbance of each sample will be measured at 340nm. The Mass Spectrophotometer will measure the light absorbance of βNADH, it will be measured at 1 minute, 4 minutes and 5 minutes to allow the change in absorbance per minute to be recorded. The results will be recorded in a Lineweaver Burke Plot, so Km and Vmax of each of the alcohols can be worked out. This will show which of the alcohols has the lowest Km. Therefore, being the best substrate for the enzyme (Schöpp. 1976). As the volume of substrate increases the change in absorbance should increase. Ethanol should have the biggest increase because it should have the smallest Km showing it will be the best substrate for the enzyme Yeast Alcohol Dehydrogenase.

Method

Before starting to the practical three solutions have to be prepared. The first is 0.1% of Bovine Serum Albumin with 10mM of Sodium Phosphate Buffer. 25ml of buffer will have 2.5mg of Bovine Serum Albumin added to it. It should be pH 7.5 and kept at 25°C until it is needed. Also a 15mM solution of b – Nicotinamide Adenine Dinucleotide is needed. 0.4317g of NAD was used instead of the 0.496g which was needed when making the βNAD solution. The Alcohol Dehydrogenase Enzyme Stock Solution will be the final solution to prepare. 15mg of solid enzyme should be diluted into 15ml of Sodium Phosphate Buffer (1ml = 1mg). Keep all solutions on ice.

Once all the solutions have been prepared, prepare the enzyme working solution by diluting 0.1ml of Alcohol Dehydrogenase Enzyme Stock Solution into 25ml of Bovine Serum Albumin Solution. Keep the solution on ice. Prepare the solutions for each cuvette by following the amounts stated in Table 1. Once the cuvette has all required elements in it, cover with parafilm and mix by inversion. Once the first cuvette is ready, add 0.1ml of the enzyme working solution to the cuvette to Assay the solution. Recover with parafilm and immediately mix the solution by inversion. Measure and record the absorbance at 340nm after 1 minute, 4 minutes and 5 minutes. Repeat this for all of the cuvettes. (Sigma Aldrich. 2013).

Buffer (ml)

b-NAD (ml)

Ethanol (ml)

Methanol (ml)

Propan-1-ol (ml)

blank

1.90

1.50

0

0

0

1

0.90

1.50

1.00

0

0

2

1.15

1.50

0.75

0

0

3

1.40

1.50

0.50

0

0

4

1.65

1.50

0.25

0

0

5

0.90

1.50

0

1.00

0

6

1.15

1.50

0

0.75

0

7

1.40

1.50

0

0.50

0

8

1.65

1.50

0

0.25

0

9

0.90

1.50

0

0

1.00

10

1.15

1.50

0

0

0.75

11

1.40

1.50

0

0

0.50

12

1.65

1.50

0

0

0.25

Table 1: list of amounts of solutions needed to be added into each cuvette.

Results

Volume of substrate (ml)

Cuvette

1 minute (AU)

4 minutes (AU)

5 minutes (AU)

Change in absorbance per minute (AU/min)

Blank 1

Blank 2

1

Ethanol – 1

- 0.019

0.290

0.380

0.090

0.75

Ethanol - 2

0.005

0.462

0.611

0.149

0.5

Ethanol - 3

0.110

-

-

0.110

0.25

Ethanol - 4

0.067

-

-

0.067

1

Methanol – 5

- 0.032

-

-0.016

0.004

0.75

Methanol – 6

- 0.036

- 0.038

- 0.015

0.023

0.5

Methanol - 7

0.014

-0.013

-0.014

-0.001

0.25

Methanol - 8

0.014

- 0.004

- 0.005

-0.001

1

Propan-1-ol – 9

-0.050

-0.055

-0.067

-0.001

0.75

Propan-1-ol - 10

-0.010

-0.042

-0.026

0.016

0.5

Propan-1-ol - 11

-0.027

0.056

0.086

0.030

0.25

Propan-1-ol - 12

0.000

0.141

0.186

0.045

Table 2: Original data from Mass Spectrophotometer

Initial Rate (Change in absorbance/min)

Alcohol added (ml)

Ethanol

Methanol

Propan-1-ol

0.25

0.067

0.008

0.016

0.50

0.11

0.015

0.030

0.75

0.149

0.023

0.045

Table 3: Data which will be used. Yellow data has been extrapolated.

As the original set of data, shown in Table 2, have some values which are incorrect. The values which are negative show that something has gone wrong with either the Mass Spectrophotometer or with the solutions which were made up. To create a graph to find out the Km of ethanol, methanol and propan-1-ol, some of the results have been omitted as they were incorrect. The data for Methanol, with 0.25ml and 0.50ml of alcohol added, has been extrapolated (Table 3). The values for ethanol increase as there is more alcohol added, this shows the enzyme works faster when it has more substrate around it (Table 3). The results for methanol and propan-1-ol also increase as there is more alcohol added to the solution. However, it is not as big an increase as ethanol, showing they are not as good a substrate for Yeast Alcohol Dehydrogenase as ethanol (Table 3).

Figure 1: Lineweaver Burke graph showing substrate specificity of Yeast Alcohol Dehydrogenase. Ethanol is the blue line, Methanol is the pink line and Propan-1-ol is the orange line.

Figure 1 shows that ethanol has a Km of 1.08mM and a Vmax of 0.356. Methanol has a Km of 6.80mM and a Vmax of 0.225 (Figure 1). Propan-1-ol has a Km of 5.51mM and a Vmax of 0.368 (Figure 1). The low Km of Ethanol shows the enzyme has a high affinity for the ethanol molecules. Yeast Alcohol Dehydrogenase has the lowest affinity for methanol because it has the highest Km value (Figure 1).

Discussion

The Lineweaver Burke Plot shows that ethanol has a Km value of 1.08mM, which is lower than the theoretical value (Figure 1). The Km value for Methanol is 6.80mM (Figure 1) which is a lot lower than the theoretical value which is 130mM. Figure 1 shows the Km of Propan-1-ol is 5.51mM, this is also much lower than the theoretical Km. Figure 1 shows that ethanol has the lowest Km and the Km values of the other two alcohols were much higher, this was expected. The change in absorbance increased as the volume of substrate increased slightly. This shows that the enzyme was working with the three alcohols but it worked best with the ethanol as it has the highest absorbance levels (Figure 1). However, the original data (Table 2) had some negative values for both methanol and propan-1-ol, so the data used in the Lineweaver Burke Plot (Figure 1) was extrapolated. As the Km of the three alcohols is very different from the theoretical values, it shows something was wrong with either the solutions or the Mass Spectrophotometer.

For this lab Yeast Alcohol Dehydrogenase catalysed the oxidation of ethanol and reduction of NAD+ (University of Arizona, 2013). It is a reaction which readily happens within the human body. Figure 1 shows the low absorbance of the alcohol at 0.25ml which shows the enzyme doesn’t work effectively when a low level of substrate is present. As the level of substrate increased the initial rate of the alcohols increased, showing the enzymes work better with a higher substrate concentration to produce an Enzyme – Substrate complex. The results of this lab are so low because the enzyme works better when it is able to reduce Acetaldehyde and oxidise NADH as it is a positive reaction and is more favourable to the enzyme (University of Arizona. 2013). Yeast Alcohol Dehydrogenase works best at room temperature but for this lab it was kept on ice until it was required. The low temperature may have inhibited the enzyme, stopping it from working as efficiently. Yeast Alcohol Dehydrogenase is only activated when sugar concentrations within the solution are low (Dickenson. 1976). If the cuvettes were contaminated from previous use, then the solution may have contained a sugar which inhibited the enzyme. The zinc atom and histidine in the active site work with the alcohol molecule to allow it to fit to the active site properly (University of Arizona. 2013). Yeast Alcohol Dehydrogenase can be inhibited by a single histidine (Leskovac. 1975). This causes the histidine and cysteine’s which are in the active site to stop working.

If this lab was to be repeated to see if the results could be improved, the same concentrations of NAD and each of the alcohols should be used. The enzyme should be kept just below room temperature to stop it from being inhibited. The cuvettes should be kept away from solutions containing sugar whilst this practical is being carried out. This should ensure the enzyme works properly.

The results show that Yeast Alcohol Dehydrogenase has group specificity for alcohols and is able to catalyse the oxidation of NAD and reduction of ethanol, methanol and propan-1-ol. However, the results are not accurate because some values gained were negative and should be repeated to see if they are correct.