Alcohol Dehydrogenase Labrapport_edit

5/25/2018 Alcohol Dehydrogenase Labrapport_edit

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Alcohol Dehydrogenase

Preparatory tasks

1. Equus caballus: 1.1.1.1: Alcohol dehydrogenaseSaccharomyces 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 theenzyme reduces acetaldehyde to ethanol during anaerobic fermentation.

4. HLADH has two domains in total and two subunits, each subunit consists of a single peptidechain 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 is149544.67 g/mol


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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]


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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 thecatalytic 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 consistentbetween the two.

10.Substrate specificityThe substrate binding pocket of HLADH seen in figure 6 and 7 consists of the amino acidsSer48, Leu57, Phe93. HLADH can take in both ethanol and pentafluorobenzyl into its


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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 thebinding 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.


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

cofactor (pink) [2]


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Laboratory tasks

1) Determine kinetic constants for alcohol dehydrogenase from yeastIn the first part of the laboratory the kinetic constants vmaxand KMfor 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 catalysedreaction NADH absorbs light at 340nm, hence the reaction speed can be analysed

spectrophotometrically. The concentration of the enzyme in each run was 6 g/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

[

+] ,

[] + max[] +

1

.

Figure 9. Primary plot


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1[] .

1

Figure 10. First secondary plot

1

[]

Figure 11. Second secondary plot


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The first secondary plots intercept is used to determine vmaxand the slope to determine KMEtOH

. From

the intercept of the second secondary plot KMNAD+

is determined.

Table 1. Summary of all substrate and enzyme concentrations in reactions

Test [Ethanol] (mM) [NAD+] (mM) [YADH] (g/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 0,2 611 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 g/ml. 50 l 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 = 1.51.5x dilution needed to make

diluted YADH stock.

600 l YADH stock (0.18 mg/ml) + 300 l Buffer = 900 l (0.12 mg/ml)

Ethanol stock solution (200 mM)

Wanted concentration of ethanol in cuvette is 2mMtotal of 100x dilution needed2 * 10x

dilutions

100 l Ethanol stock(200mM) + 900 l Buffer = 1 ml diluted ethanol stock (20mM)

NAD+stock solution (20 mM)

Wanted concentration of NAD+in cuvette is 0.05 mMtotal of 400x dilution needed2* 20x

dilutions.

100 l NAD+stock (20mM) + 1900 l Buffer = 2 ml diluted NAD

+stock (1 mM)


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Table 2. Recipe test 1

Substance Stock solution[mM] Diluted

solution[mM]

Volume [l] Concentration in

cuvette[mM]

NAD+ 20 1 50 0,05

Ethanol 200 20 100 2YADH 0,18 mg/ml 0,12 mg/ml 50 0,006 mg/ml

Buffer 800



solution[mM]


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/mlBuffer 800



solution[mM]


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



solution[mM]


cuvette[mM]

NAD+ 20 20 50 1

Ethanol 200 20 100 2


Buffer 800



solution[mM]


cuvette[mM]

NAD+ 20 1 50 0,05

Ethanol 200 40 100 4


Buffer 800


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solution[mM]


cuvette[mM]

NAD+ 20 4 50 0,2


Buffer 800



solution[mM]


cuvette[mM]

NAD+ 20 10 50 0,5

Ethanol 200 40 100 4


Buffer 800



solution[mM]


cuvette[mM]

NAD+ 20 20 50 1

Ethanol 200 40 100 4


Buffer 800



solution[mM]


cuvette[mM]

NAD+ 20 1 50 0,05

Ethanol 200 50 100 5


Buffer 800



solution[mM]


cuvette[mM]

NAD+ 20 4 50 0,2

Ethanol 200 50 100 5


Buffer 800


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solution[mM]


cuvette[mM]

NAD+ 20 10 50 0,5


Buffer 800



solution[mM]


cuvette[mM]

NAD+ 20 20 50 1

Ethanol 200 50 100 5


Buffer 800



solution[mM]


cuvette[mM]

NAD+ 20 1 50 0,05

Ethanol 200 80 100 8


Buffer 800


Substance Stock

solution[mM]

Diluted

solution[mM]


cuvette[mM]

NAD+ 20 4 50 0,2

Ethanol 200 80 100 8


Buffer 800



solution[mM]


cuvette[mM]

NAD+ 20 10 50 0,5

Ethanol 200 80 100 8


Buffer 800


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solution[mM]


cuvette[mM]

NAD+ 20 20 50 0,5


Buffer 800

Results

Table 18. Measurements for the different cuvettes and their rates

Tes

t

[Ethanol]

(mM)

[NAD+]

(mM)

[YADH]

(g/ml) A (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 54 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 11,3636363614 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


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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

R = 0,9999

y = 0,9079x + 1,0234

R = 0,9994

y = 0,6699x + 1,559

R = 0,9996

y = 0,4955x + 1,4806

R = 0,9994

0

5

10

15

20

25

30

35

40

0 5 10 15 20 25

1/v(min)

1/[NAD+] (mM-1)

Primary plot2 mM Ethanol

4 mM Ethanol

5 mM Ethanol

8 mM Ethanol

y = 1,4031x + 1,1255

R = 0,3617

0

0,5

1

1,5

2

2,5

0 0,1 0,2 0,3 0,4 0,5 0,6

Intercept(min)

1/[Ethanol] (mM-1)

Secondary plot (intercept)


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Table 20. Values used for second secondary plot, retrieved from figure 12.

[Ethanol] (mM) Slope (minmM) 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 aFigure 13 gives 1= 1,1255 min while the slope gives

= 1,4031 minmM.With this is calculated to 11,1255 = 0,888 min-1and to1,4031 0,888 1 = 1,246mM.The more common unit for is U/mg and in order to convert the velocity, Lambert-Beers law isused.

Lambert-Beers law: = =

= 6,2

1

1

= 0,888 min11 6.2 11 = 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

R = 0,9933

0

0,5

1

1,5

2

0 0,1 0,2 0,3 0,4 0,5 0,6

Slope(m

in*mM)

1/[Ethanol] (mM^-1)

Secondary plot (slope)


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The intercept inFigure 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 fromhorse 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 kcat/KM.

= [] + []1

=max[] +

1

By plotting 1/v vs 1/[S] for each reaction a line with the intercept1

and slope is given.

Dilution calculations

HLADH stock solution(26 mg/ml)

Concentration of enzyme in cuvette is 2.6 mg/ml. 100 l from diluted stock is put in cuvette, thisgives a dilution of 10x. Since the HLADH original stock solution can be used there is no need to make

a diluted HLADH stock.

100 l HLADH stock (26 mg/ml) + 900 l Buffer = 1 ml (2.6 mg/ml)

Ethanol stock solution (200 mM)

Wanted concentration of ethanol in cuvette is 2mMtotal of 100x dilution needed5x * 20x

dilutions are done.

200 l Ethanol stock (200mM) + 800 l Buffer = 1 ml diluted ethanol stock (40mM)

Hexanol stock solution (10 mM)

Wanted concentration of Hexanol in cuvette is 0.5 mMtotal of 20x dilution needed10x * 2x

dilutions are done.

200 l Hexanol stock (20mM) + 1800 l Buffer = 2 ml diluted Hexanol stock (1 mM)


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Table 22. Recipe test 1 Ethanol and YADH


solution[mM]


cuvette[mM]

NAD+ 20 20 50 1

Ethanol 200 10 50 0,5YADH 0,18 mg/ml 0,06 mg/ml 50 0,003 mg/ml

Buffer 850



solution[mM]


cuvette[mM]

NAD+ 20 20 50 1

Ethanol 200 40 50 2


Buffer 850



solution[mM]


cuvette[mM]

NAD+ 20 20 50 1

Ethanol 200 100 50 5


Buffer 850



solution[mM]


cuvette[mM]

NAD+ 20 20 50 1

Ethanol 200 200 50 10


Buffer 850

Table 26. Recipe test 1 Ethanol and HLADH

Substance Stock

solution[mM]

Diluted

solution[mM]


cuvette[mM]

NAD+ 20 20 50 1

Ethanol 10 10 50 0,5

HLADH 26 mg/ml - 50 1,3 mg/ml

Buffer 850


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Substance Stock

solution[mM]

Diluted

solution[mM]


cuvette[mM]

NAD+ 20 20 50 1

Ethanol 10 40 50 2HLADH 26 mg/ml - 50 1,3 mg/ml

Buffer 850


Substance Stock

solution[mM]

Diluted

solution[mM]


cuvette[mM]

NAD+ 20 20 50 1

Ethanol 10 100 50 5

HLADH 26 mg/ml - 50 1,3 mg/mlBuffer 850


Substance Stock

solution[mM]

Diluted

solution[mM]


cuvette[mM]

NAD+ 20 20 50 1

Ethanol 10 200 50 10


Buffer 850

Table 30. Recipe test 1 Hexanol and YADH


solution[mM]


cuvette[mM]

NAD+ 20 - 50 1

Hexanol 10 1 500 0,5

YADH 0,18 mg/ml - 333 0,06 mg/ml

Buffer 117



solution[mM]


cuvette[mM]

NAD+ 20 20 50 1

Hexanol 10 4 500 2

YADH 0,18 mg/ml - 333 0,06 mg/ml

Buffer 117


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solution[mM]


cuvette[mM]

NAD+ 20 - 50 1

Hexanol 10 8 500 4YADH 0,18 mg/ml - 333 0,06 mg/ml

Buffer 117



solution[mM]


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


solution[mM]


cuvette[mM]

NAD+ 20 - 50 1

Hexanol 10 1 500 0,5


Buffer 350



solution[mM]


cuvette[mM]

NAD+ 20 - 50 1

Hexanol 10 4 500 2


Buffer 350



solution[mM]


cuvette[mM]

NAD+ 20 - 50 1

Hexanol 10 8 500 4


Buffer 350


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solution[mM]


cuvette[mM]

NAD+ 20 - 50 1

Hexanol 10 - 500 5HLADH 26 mg/ml - 100 2,6 mg/ml

Buffer 350

Results part 2

Table 38. Values for YADH 3 g/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 = 11,7963 = 0,557 1.

= 0.557 1

1 6.2 11 = 0,090 /

= 0,090 1 = 0,090 = 0,090

= 3

= 3

1 = 3

y = 13,766x + 1,7963

R = 0,9994

0

5

10

15

20

25

30

35

0 0,5 1 1,5 2 2,5

1/v(min)

1/[Ethanol] (mM-1)

YADH: Ethanol


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= 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

// = 8,024 105

= [] =(0,09/60)

8,024 105/ = 18,69 1

=18,69 17,668 = 2,4411

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 = 12,0957 = 0,477 1.

= 0.477 1

1

6.2

11= 0,077 /

y = 1,2722x + 2,0957

R = 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(min)

1/[Ethanol] (mM-1)

HLADH: Ethanol


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= 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

() = 40693,86Since 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 10,607 = 0,066 11

Table 40. Values for YADH 60 g/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 g/ml with varying hexanol concentrations.

y = 21,877x + 1,6637

R = 0,9999

0

5

10

15

20

25

30

35

40

45

50

0 0,5 1 1,5 2 2,5

1/v

(min)

1/[Hexanol] (/mM)

YADH: Hexanol


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The Intercept =1

= 1,6637 min gives = 11,6637 = 0,601 1.

= 0.601 1

1 6.2 11 = 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 11Table 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


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

The Intercept =1

= 1,6688 min gives = 11,6688 = 0,599 1.

= 0.599 1

1 6.2 11 = 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 10,475 = 0,053 11

y = 0,7931x + 1,6688

R = 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(min)

1/[Hexanol] (/mM)

HLADH: Hexanol


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Table 42. Kinetic specificity constants for YADH and HLADH with ethanol and hexanol substrates

YADH: Ethanol

Kcat/KM (mM

-1

s

-1

)

YADH: Hexanol

Kcat/KM (mM

-1

s

-1

)

HLADH: Ethanol

Kcat/KM(mM

-1

s

1

)

HLADH: Hexanol

Kcat/KM (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.


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References

[1] RSCB PDB. [Online].

Available:http://www.rcsb.org/pdb/explore.do?structureId=1ADB[Cited 02 12 2013].


Available:http://www.rcsb.org/pdb/explore.do?structureId=2HCY[Cited 02 12 2013].


Available:http://www.rcsb.org/pdb/explore.do?structureId=6adh[Cited 02 12 2013].


Available:http://www.rcsb.org/pdb/explore.do?structureId=8adh [Cited 02 12 2013].


Available:http://www.rcsb.org/pdb/explore.do?structureId=1hld [Cited 02 12 2013].
http://www.rcsb.org/pdb/explore.do?structureId=1ADBhttp://www.rcsb.org/pdb/explore.do?structureId=1ADBhttp://www.rcsb.org/pdb/explore.do?structureId=1ADBhttp://www.rcsb.org/pdb/explore.do?structureId=2HCYhttp://www.rcsb.org/pdb/explore.do?structureId=2HCYhttp://www.rcsb.org/pdb/explore.do?structureId=2HCYhttp://www.rcsb.org/pdb/explore.do?structureId=6adhhttp://www.rcsb.org/pdb/explore.do?structureId=6adhhttp://www.rcsb.org/pdb/explore.do?structureId=6adhhttp://www.rcsb.org/pdb/explore.do?structureId=http://www.rcsb.org/pdb/explore.do?structureId=http://www.rcsb.org/pdb/explore.do?structureId=http://www.rcsb.org/pdb/explore.do?structureId=http://www.rcsb.org/pdb/explore.do?structureId=http://www.rcsb.org/pdb/explore.do?structureId=http://www.rcsb.org/pdb/explore.do?structureId=http://www.rcsb.org/pdb/explore.do?structureId=http://www.rcsb.org/pdb/explore.do?structureId=6adhhttp://www.rcsb.org/pdb/explore.do?structureId=2HCYhttp://www.rcsb.org/pdb/explore.do?structureId=1ADB

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Alcohol Dehydrogenase Labrapport_edit