Alcohol Dehydrogenase

Preparatory tasks

1. Equus caballus: 1.1.1.1: Alcohol dehydrogenase
Saccharomyces cerevisiae: 1.1.1.1: Alcohol dehydrogenase and 1.1.1.2: Alcohol
dehydrogenase.
2.
Figure 1. The reaction catalyzed by alcohol dehydrogenase in structure formulas
3. The function of HLADH is to degrade ethanol which is a toxic molecule. In YADH however the
enzyme reduces acetaldehyde to ethanol during anaerobic fermentation.
4. HLADH has two domains in total and two subunits, each subunit consists of a single peptide
chain of 375 amino acids.
YADH has two domains in total and four subunits, each subunit consists of a single peptide
chain of 348 amino acids.
5. The molecular weight of HLADH is : 81387,72 g/mol,and the molecular weight of YADH is
149544.67 g/mol
6.
Figure 2. Structure of HLADH showing the active site consisting of Cys174, His67, Cys46 and
Ser48 and zinc atoms in black [1]
7.
Figure 3. Structure of YADH with the subunits in different colors and zinc atoms in grey. [2]

Figure 4. Structure of HLADH with cofactor bound (pink) and unbound (green) [3][4]
8. The function of the conformational change of 10° is to move the bound cofactor closer to the
catalytic site. The cofactor does not move itself but the enzyme changes conformation which
moves the catalytic site closer to the cofactor. Since the cofactor has to bind first, it gives the
information that the mechanism is ordered and NAD
+
needs to bind before the substrate.


Figure 5. HLADH active site structure, zinc atom in grey [1]
9. By comparing figure 5 to the Fersht-hand out it can be seen that the structures are consistent
between the two.
10. Substrate specificity
The substrate binding pocket of HLADH seen in figure 6 and 7 consists of the amino acids
Ser48, Leu57, Phe93. HLADH can take in both ethanol and pentafluorobenzyl into its
substrate binding pocket, using pentafluorobenzyl as a substitute for hexanol it can be said
that both ethanol and hexanol fit well into the binding pocket of HLADH.

Figure 6. Substrate binding pocket of HLADH (green), the substrate ethanol (Red), zinc atom (grey)
and cofactor (pink) [1]


Figure 7. Substrate binding pocket of HLADH (green), the substrate Pentafluorobenzyl (Red), zinc
atom (grey) and cofactor (pink) [5]
The substrate binding pocket in YADH differs from that in HLADH, in YADH it consists of Thr45, Trp54
and Trp92. Comparing figure 8 to figure 6/7 it can be seen that the amino acids building up the
binding pocket are much bulkier in YADH. Using this information a hypothesis can be formed, that
YADH possibly will have higher activity to ethanol compared to hexanol due to steric hindrance.


Figure 8. Substrate binding pocket of YADH (green), the substrate ethanol (Red), zinc atom (grey) and
cofactor (pink) [2]

Laboratory tasks
1) Determine kinetic constants for alcohol dehydrogenase from yeast
In the first part of the laboratory the kinetic constants v
max
and K
M
for Yeast alcohol dehydrogenase
with the two substrates NAD
+
and ethanol. To do this four different concentrations of each substrate
was measured, resulting in 16 different reactions. The product of the enzymatically catalysed
reaction NADH absorbs light at 340nm, hence the reaction speed can be analysed
spectrophotometrically. The concentration of the enzyme in each run was 6 µg/ml.
The kinetic mechanism of alcohol dehydrogenase is classified as a bi bi compulsory ordered reaction,
by using this information the rate equation is given as follows:
=


[
+
][]

+

+

[
+
] +

+
[] +[
+
][]

1

=

+
+

[
+
][]
+

[
+
]

[
+
][]
+

+
[]

[
+
][]
+
[
+
][]

[
+
][]

1

=

+

[
+
][]
+

[]
+

+

[
+
]
+
1

1

=
1
[
+
]

+

[]
+

+

� +

max
[]
+
1

1

1
[
+
]
ℎ ℎ , ℎ ℎ

+

[]
+

+

� ℎ

max
[]
+
1

. ℎ ℎ

Figure 9. Primary plot

ℎ ℎ ℎ
1
[]
ℎ .
ℎ ℎ


1

Figure 10. First secondary plot
ℎ ℎ ℎ
1
[]

ℎ ℎ

+

+

Figure 11. Second secondary plot

The first secondary plots intercept is used to determine v
max
and the slope to determine K
M
EtOH
. From
the intercept of the second secondary plot K
M
NAD+
is determined.
Table 1. Summary of all substrate and enzyme concentrations in reactions
Test [Ethanol] (mM) [NAD+] (mM) [YADH] (µg/ml)
1 2 0,05 6
2 2 0,2 6
3 2 0,5 6
4 2 1 6
5 4 0,05 6
6 4 0,2 6
7 4 0,5 6
8 4 1 6
9 5 0,05 6
10 5 0,2 6
11 5 0,5 6
12 5 1 6
13 8 0,05 6
14 8 0,2 6
15 8 0,5 6
16 8 1 6

Dilution calculations
YADH stock solution(0.18 mg/ml)
Concentration of enzyme in cuvette is 6 µg/ml. 50 µl from diluted stock is put in cuvette, this gives a
dilution of 20x. 20 * 0.006 mg/ml = 0.12 mg/ml  0.18/0.12 = 1.5  1.5x dilution needed to make
diluted YADH stock.
600 µl YADH stock (0.18 mg/ml) + 300 µl Buffer = 900 µl (0.12 mg/ml)
Ethanol stock solution (200 mM)
Wanted concentration of ethanol in cuvette is 2mM  total of 100x dilution needed  2 * 10x
dilutions
100 µl Ethanol stock(200mM) + 900 µl Buffer = 1 ml diluted ethanol stock (20mM)
NAD
+
stock solution (20 mM)
Wanted concentration of NAD
+
in cuvette is 0.05 mM  total of 400x dilution needed  2* 20x
dilutions.
100 µl NAD
+
stock (20mM) + 1900 µl Buffer = 2 ml diluted NAD
+
stock (1 mM)

Table 2. Recipe test 1
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 1 50 0,05
Ethanol 200 20 100 2
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800

Table 3. Recipe test 2
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 4 50 0,2
Ethanol 200 20 100 2
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800

Table 4. Recipe test 3
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 10 50 0,5
Ethanol 200 20 100 2
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800

Table 5. Recipe test 4
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 20 50 1
Ethanol 200 20 100 2
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800

Table 6. Recipe test 5
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 1 50 0,05
Ethanol 200 40 100 4
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800


Table 7. Recipe test 6
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 4 50 0,2
Ethanol 200 40 100 4
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800

Table 8. Recipe test 7
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 10 50 0,5
Ethanol 200 40 100 4
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800


Table 9. Recipe test 8
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 20 50 1
Ethanol 200 40 100 4
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800

Table 10. Recipe test 9
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 1 50 0,05
Ethanol 200 50 100 5
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800

Table 11. Recipe test 10
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 4 50 0,2
Ethanol 200 50 100 5
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800


Table 12. Recipe test 11
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 10 50 0,5
Ethanol 200 50 100 5
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800
Table 13. Recipe test 12
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 20 50 1
Ethanol 200 50 100 5
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800

Table 14. Recipe test 13
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 1 50 0,05
Ethanol 200 80 100 8
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800

Table 15. Recipe test 14
Substance Stock
solution[mM]
Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 4 50 0,2
Ethanol 200 80 100 8
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800

Table 16. Recipe test 15
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 10 50 0,5
Ethanol 200 80 100 8
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800



Table 17. Recipe test 16
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD
+
20 20 50 0,5
Ethanol 200 80 100 8
YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml
Buffer 800
Results
Table 18. Measurements for the different cuvettes and their rates
Tes
t
[Ethanol]
(mM)
[NAD+]
(mM)
[YADH]
(µg/ml) ΔA (min
-1
) 1/[NAD+] (mM
-1
) 1/v (min)
1 2 0,05 6 0,03 20 33,33333333
2 2 0,2 6 0,1 5 10
3 2 0,5 6 0,2 2 5
4 2 1 6 0,29 1 3,448275862
5 4 0,05 6 0,052 20 19,23076923
6 4 0,2 6 0,187 5 5,347593583
7 4 0,5 6 0,362 2 2,762430939
8 4 1 6 0,46 1 2,173913043
9 5 0,05 6 0,067 20 14,92537313
10 5 0,2 6 0,197 5 5,076142132
11 5 0,5 6 0,355 2 2,816901408
12 5 1 6 0,46 1 2,173913043
13 8 0,05 6 0,088 20 11,36363636
14 8 0,2 6 0,245 5 4,081632653
15 8 0,5 6 0,4 2 2,5
16 8 1 6 0,54 1 1,851851852


Figure 12. Primary plot
Table 19. values used for first secondary plot retrieved from figure 12.
[Ethanol] (mM) Intercept (min) 1/[Ethanol] (mM
-1
)
2 1,9475 0,5
4 1,0234 0,25
5 1,559 0,2
8 1,4806 0,125


Figure 13. First secondary plot
y = 1,5711x + 1,9475
R² = 0,9999
y = 0,9079x + 1,0234
R² = 0,9994
y = 0,6699x + 1,559
R² = 0,9996
y = 0,4955x + 1,4806
R² = 0,9994
0
5
10
15
20
25
30
35
40
0 5 10 15 20 25
1
/
v

(
m
i
n
)

1/[NAD+] (mM
-1
)
Primary plot
2 mM Ethanol
4 mM Ethanol
5 mM Ethanol
8 mM Ethanol
y = 1,4031x + 1,1255
R² = 0,3617
0
0,5
1
1,5
2
2,5
0 0,1 0,2 0,3 0,4 0,5 0,6
I
n
t
e
r
c
e
p
t

(
m
i
n
)

1/[Ethanol] (mM
-1
)
Secondary plot (intercept)
Table 20. Values used for second secondary plot, retrieved from figure 12.
[Ethanol] (mM) Slope (min·mM) 1/[Ethanol] (mM
-1
)
2 1,5711 0,5
4 0,9079 0,25
5 0,6699 0,2
8 0,4955 0,125


Figure 14. Second secondary plot
The intercept in a Figure 13 gives
1

= 1,1255 min while the slope gives

= 1,4031 min·mM.
With this

is calculated to
1
1,1255
= 0,888 min
-1
and

to
1,4031 ∙ ∙ 0,888
−1
= 1,246 mM.
The more common unit for

is U/mg and in order to convert the velocity, Lambert-Beers law is
used.
Lambert-Beers law: = ∙ ∙ → =


= 6,2
−1

−1

=
0,888 min
−1
1 ∙ 6.2
−1

−1
= 0,143 /
Volume = 1 ml

= 0,143

∙ 1 = 0,143

= 0,143

= 6

= 6 = 0,006

=
0,143
0,006
≈ 23,83 /
y = 2,8909x + 0,1342
R² = 0,9933
0
0,5
1
1,5
2
0 0,1 0,2 0,3 0,4 0,5 0,6
S
l
o
p
e

(
m
i
n
*
m
M
)

1/[Ethanol] (mM^-1)
Secondary plot (slope)
The intercept in Figure 14

+

= 0,1342 ∙ gives

+
= 0,1342 ∙ ∙
0,888
−1
= 0,119
Table 21. Calculated kinetic constants

(U/mg)

(mM)

+
(mM)
23,83 1,246 0,119

2) Compare substrate specificity for ethanol and hexanol for alcohol dehydrogenase from
horse liver and yeast.
To examine the specificity of alcohol dehydrogenase from horse liver and yeast to the two substrates
ethanol and hexanol, four different reactions were measured. For each of these reactions four
different substrate concentrations were measured. The reactions measured were for HLADH with
Hexanol/Ethanol and YADH with Hexanol/Ethanol. The specificity is defined as k
cat
/K
M.

=

[]

+[]

1

=

max[]
+
1

By plotting 1/v vs 1/[S] for each reaction a line with the intercept
1

and slope

is given.
Dilution calculations
HLADH stock solution(26 mg/ml)
Concentration of enzyme in cuvette is 2.6 mg/ml. 100 µl from diluted stock is put in cuvette, this
gives a dilution of 10x. Since the HLADH original stock solution can be used there is no need to make
a diluted HLADH stock.
100 µl HLADH stock (26 mg/ml) + 900 µl Buffer = 1 ml (2.6 mg/ml)
Ethanol stock solution (200 mM)
Wanted concentration of ethanol in cuvette is 2mM  total of 100x dilution needed  5x * 20x
dilutions are done.
200 µl Ethanol stock (200mM) + 800 µl Buffer = 1 ml diluted ethanol stock (40mM)
Hexanol stock solution (10 mM)
Wanted concentration of Hexanol in cuvette is 0.5 mM  total of 20x dilution needed  10x * 2x
dilutions are done.
200 µl Hexanol stock (20mM) + 1800 µl Buffer = 2 ml diluted Hexanol stock (1 mM)

Table 22. Recipe test 1 Ethanol and YADH
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 20 50 1
Ethanol 200 10 50 0,5
YADH 0,18 mg/ml 0,06 mg/ml 50 0,003 mg/ml
Buffer 850
Table 23. Recipe test 2 Ethanol and YADH
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 20 50 1
Ethanol 200 40 50 2
YADH 0,18 mg/ml 0,06 mg/ml 50 0,003 mg/ml
Buffer 850

Table 24. Recipe test 3 Ethanol and YADH
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 20 50 1
Ethanol 200 100 50 5
YADH 0,18 mg/ml 0,06 mg/ml 50 0,003 mg/ml
Buffer 850

Table 25. Recipe test 4 Ethanol and YADH
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 20 50 1
Ethanol 200 200 50 10
YADH 0,18 mg/ml 0,06 mg/ml 50 0,003 mg/ml
Buffer 850

Table 26. Recipe test 1 Ethanol and HLADH
Substance Stock
solution[mM]
Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 20 50 1
Ethanol 10 10 50 0,5
HLADH 26 mg/ml - 50 1,3 mg/ml
Buffer 850



Table 27. Recipe test 2 Ethanol and HLADH
Substance Stock
solution[mM]
Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 20 50 1
Ethanol 10 40 50 2
HLADH 26 mg/ml - 50 1,3 mg/ml
Buffer 850

Table 28. Recipe test 3 Ethanol and HLADH
Substance Stock
solution[mM]
Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 20 50 1
Ethanol 10 100 50 5
HLADH 26 mg/ml - 50 1,3 mg/ml
Buffer 850

Table 29. Recipe test 4 Ethanol and HLADH
Substance Stock
solution[mM]
Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 20 50 1
Ethanol 10 200 50 10
HLADH 26 mg/ml - 50 1,3 mg/ml
Buffer 850

Table 30. Recipe test 1 Hexanol and YADH
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 - 50 1
Hexanol 10 1 500 0,5
YADH 0,18 mg/ml - 333 0,06 mg/ml
Buffer 117

Table 31. Recipe test 2 Hexanol and YADH
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 20 50 1
Hexanol 10 4 500 2
YADH 0,18 mg/ml - 333 0,06 mg/ml
Buffer 117


Table 32. Recipe test 3 Hexanol and YADH
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 - 50 1
Hexanol 10 8 500 4
YADH 0,18 mg/ml - 333 0,06 mg/ml
Buffer 117

Table 33. Recipe test 4 Hexanol and YADH
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 - 50 1
Hexanol 10 - 500 5
YADH 0,18 mg/ml - 333 0,06 mg/ml
Buffer 117

Table 34. Recipe test 1 Hexanol and HLADH
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 - 50 1
Hexanol 10 1 500 0,5
HLADH 26 mg/ml - 100 2,6 mg/ml
Buffer 350

Table 35. Recipe test 2 Hexanol and HLADH
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 - 50 1
Hexanol 10 4 500 2
HLADH 26 mg/ml - 100 2,6 mg/ml
Buffer 350

Table 36. Recipe test 3 Hexanol and HLADH
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 - 50 1
Hexanol 10 8 500 4
HLADH 26 mg/ml - 100 2,6 mg/ml
Buffer 350

Table 37. Recipe test 4 Hexanol and HLADH
Substance Stock solution[mM] Diluted
solution[mM]
Volume [µl] Concentration in
cuvette[mM]
NAD+ 20 - 50 1
Hexanol 10 - 500 5
HLADH 26 mg/ml - 100 2,6 mg/ml
Buffer 350

Results part 2
Table 38. Values for YADH 3 µg/ml with varying ethanol concentrations
[Ethanol] (mM) Delta abosorbance (min
-1
) 1/v (min) 1/[Ethanol] (mM
-1
)
0,5 0,034 29,41176471 2
2 0,121 8,26446281 0,5
5 0,216 4,62962963 0,2
10 0,292 3,424657534 0,1


Figure 15. Kinetic plot for YADH with varying ethanol concentrations
The Intercept =
1

= 1,7963 min gives

=
1
1,7963
= 0,557
−1
.

=
0.557
−1
1 ∙ 6.2
−1

−1
= 0,090 /

= 0,090

∙ 1 = 0,090

= 0,090

= 3

= 3

∗ 1 = 3
y = 13,766x + 1,7963
R² = 0,9994
0
5
10
15
20
25
30
35
0 0,5 1 1,5 2 2,5
1
/
v

(
m
i
n
)

1/[Ethanol] (mM
-1
)
YADH: Ethanol

=
0,090
0,003
= 30

Slope =

= 13,766 ∙ →

= 13,766 ∙ ∙ 0,557
−1
= 7,668

( ) = 37386,17

Since each subunit has an own active site.
[] = 3

=
0,003
37386,17
/
/
= 8,024 ∙ 10
−5

=

[]
=
(0,09/60)
8,024 ∙ 10
−5
/

= 18,69
−1

=
18,69
−1
7,668
= 2,44
−1

−1

Table 39. Values for HLADH 1.3 mg/ml with varying ethanol concentrations
[Ethanol] (mM) Delta abosorbance (min
-1
) 1/v (min) 1/[Ethanol] (mM
-1
)
0,5 0,22 4,545454545 2
2 0,31 3,225806452 0,5
5 0,46 2,173913043 0,2
10 0,5 2 0,1


Figure 16. Kinetic plot for HLADH 1.3 mg/ml with varying ethanol concentrations
The Intercept =
1

= 2,0957 min gives

=
1
2,0957
= 0,477
−1
.

=
0.477
−1
1 ∙ 6.2
−1

−1
= 0,077 /
y = 1,2722x + 2,0957
R² = 0,919
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
0 0,5 1 1,5 2 2,5
1
/
v

(
m
i
n
)

1/[Ethanol] (mM
-1
)
HLADH: Ethanol

= 0,077

∙ 1 = 0,077

= 0,077

= 1,3

= 1,3

∗ 1 = 1,3

=
0,077
1,3
= 0,059

Slope =

= 1,2722 ∙ →

= 1,2722 ∙ ∙ 0,477
−1
= 0,607

( ) = 40693,86

Since each subunit has an own active site.
[] = 1,3

=
1,3
40693,86
/
/
= 0,0320

=

[]
=
(0,077/60)
0,0320
/

= 0,0401
−1

=
0,0401
−1
0,607
= 0,066
−1

−1

Table 40. Values for YADH 60 µg/ml with varying hexanol concentrations
[Hexanol] (mM) Delta absorbance (min
-1
) 1/v (min) 1/[Hexanol] (mM
-1
)
0,5 0,022 45,45454545 2
2 0,081 12,34567901 0,5
4 0,136 7,352941176 0,25


Figure 17. Kinetic plot for YADH 60 µg/ml with varying hexanol concentrations.

y = 21,877x + 1,6637
R² = 0,9999
0
5
10
15
20
25
30
35
40
45
50
0 0,5 1 1,5 2 2,5
1
/
v

(
m
i
n
)

1/[Hexanol] (/mM)
YADH: Hexanol
The Intercept =
1

= 1,6637 min gives

=
1
1,6637
= 0,601
−1
.

=
0.601
−1
1 ∙ 6.2
−1

−1
= 0,097 /

= 0,097

∙ 1 = 0,097

= 0,097

= 60

= 60

∗ 1 = 60

=
0,097
0,06
= 1,62

Slope =

= 21,877 ∙ →

= 21,877 ∙ ∙ 0,601
−1
= 13,15

( ) = 37386,17

Since each subunit has an own active site.
[] = 60

=
0,06
37386,17
/
/
= 0,0016

=

[]
=
(
0,097
60
)
0,0016
/

= 1,01
−1

=
1,01
−1
13,15
= 0,077
−1

−1

Table 41. Values for HLADH 2.6 mg/ml with varying hexanol concentrations
[Hexanol] (mM) Delta abosorbance (min
-1
) 1/v (min) 1/[Hexanol] (mM
-1
)
0,5 0,313 3,194888179 2
2 0,415 2,409638554 0,5
4 0,51 1,960784314 0,25
5 0,69 1,449275362 0,2


Figure 18. Kinetic plot for HLADH 2.6 mg/ml with varying hexanol concentrations.

The Intercept =
1

= 1,6688 min gives

=
1
1,6688
= 0,599
−1
.

=
0.599
−1
1 ∙ 6.2
−1

−1
= 0,097 /

= 0,097

∙ 1 = 0,097

= 0,097

= 2,6

= 2,6

∗ 1 = 2,6

=
0,097
2,6
= 0,037

Slope =

= 0,7931 ∙ →

= 0,7931 ∙ ∙ 0,599
−1
= 0,475

( ) = 40693,86

Since each subunit has an own active site.
[] = 2,6

=
2,6
40693,86
/
/
= 0,0639

=

[]
=
(
0,097
60
)
0,0639
/

= 0,025
−1

=
0,025
−1
0,475
= 0,053
−1

−1


y = 0,7931x + 1,6688
R² = 0,8333
0
0,5
1
1,5
2
2,5
3
3,5
0 0,5 1 1,5 2 2,5
1
/
v

(
m
i
n
)

1/[Hexanol] (/mM)
HLADH: Hexanol

Table 42. Kinetic specificity constants for YADH and HLADH with ethanol and hexanol substrates
YADH: Ethanol
K
cat
/K
M
(mM
-1
s
-1
)
YADH: Hexanol
K
cat
/K
M
(mM
-1
s
-1
)
HLADH: Ethanol
K
cat
/K
M
(mM
-1
s
1
)
HLADH: Hexanol
K
cat
/K
M
(mM
1
s
-1
)
2,44 0,077 0,066 0,053

Discussion
The hypothesis saying that YADH has higher specificity for ethanol than hexanol is confirmed by the
results presented in table 42. Also HLADH shows higher specificity for ethanol than hexanol aswell,
which indicates that ethanol fits better in the substrate binding spot. The reason to why YADH has
higher specificity for ethanol is due to the bulkier amino acids in the binding spot, the steric
hindrance caused by these makes it easier for ethanol to bind than hexanol.
To improve the results and reduce the errors in these tests duplicates could have been made,
however since this would have increased the laboration time needed and the amount of enzyme it
was not possible to do so.

References

[1] RSCB PDB. [Online].
Available: http://www.rcsb.org/pdb/explore.do?structureId=1ADB [Cited 02 12 2013].
[2] RSCB PDB. [Online].
Available: http://www.rcsb.org/pdb/explore.do?structureId=2HCY [Cited 02 12 2013].
[3] RSCB PDB. [Online].
Available: http://www.rcsb.org/pdb/explore.do?structureId=6adh [Cited 02 12 2013].
[4] RSCB PDB. [Online].
Available: http://www.rcsb.org/pdb/explore.do?structureId=8adh [Cited 02 12 2013].
[5] RSCB PDB. [Online].
Available: http://www.rcsb.org/pdb/explore.do?structureId=1hld [Cited 02 12 2013].