Alcohol Dehydrogenase

ALCOHOL DEHYDROGENASE A comprehensive overview and

experimental analysis

Elliott Weideman

ALCOHOL DEHYDROGENASE

What is it? ADH Enzyme that breaks down alcohols

Why is it important? In most animals it is used to metabolize

alcohols that have been ingested. Yeasts and bacteria use ADH in reverse to

convert sugars into ethanol via fermentation.


RCH2OH + NAD+

RCHO + NADH + H+

Chemical Reaction

ADH

NAD+ is needed as an important cofactor for ADH

Experimental Question What is the rate of reaction that ADH is capable

of maintaining?

Experimental Design Overview Chemical assay techniques using

spectrophotometry to measure the amount of product synthesized from ADH.


Materials Yeast ADH - Provided from Sigma NAD+ - Provided from Sigma 95% Ethanol - Provided from Sigma Tris Buffer pH 8 - Provided from AcrosEquipment Genesys 10 UV/vis Spectrophotometer - Provided

from ThermoSpectronic Cuvettes - Provided from Fisher Scientific

ALCOHOL DEHYDROGENASES. Cerevisiae

Procedure Obtain Tris buffer - pH 8 Prepare ADH enzyme by adding dry reagent with

water to equal 20 units/mL Dilute Ethanol to concentrations of 0.7M, 0.375M, 0.1M, 0.5M and 0.25M

C1 V1 =C2 V2

Prepare NAD+ to final concentration of 15mM


Procedure Enzyme assays by combining the following

900uL Tris buffer 33uL NAD+ 33uL ethanol (0.7M, 0.375M, 0.1M, 0.05M, and 0.025M) 33uL ADH

The solution was mixed thoroughly and gently before quickly being measured in the spectrophotometer at 340nm


Procedure Assays were measured for absorbance at T0 and

T1 Average rate (Abs340 T1 – Abs340T0/min) was recorded

Three trials were run for each [ethanol]


Data

ALCOHOL DEHYDROGENASE[Ethanol] M Abs T0 Abs T1

Rate (ΔA340 /min)

Average Rate

0.73+ 3+ -

0.082+2.923 3+ 0.080+2.917 3+ 0.083+

0.3752.466 3+ 0.534+

0.539+1.072 1.102 0.0301.946 3+ 1.054+

0.11.066 2.182 1.116

1.0031.215 2.229 1.0141.283 2.163 0.880

0.050.904 1.443 0.539

0.5520.854 1.47 0.6160.697 1.197 0.500

0.0250.651 1.152 0.501

0.4350.566 0.928 0.3620.636 1.077 0.441

Results Lineweaver-Burke Data and Plot


1/[Ethanol] 1/Abs T1

1.4285 0.539539

2.6667 0.617408

10 0.84685

20 1.13729

40 1.62507

0 5 10 15 20 25 30 35 40 450

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

f(x) = 0.0276960846636675 x + 0.542802013525725R² = 0.993762636749275

1/[Ethanol] M

1/A

bsor

banc

e 34

0nm

Results Vmax – The fastest the enzyme can perform

under ideal circumstances 0.0511 M/min

Km – Michaelis constant (several rate constants) 1.8423 mM


3D rendering of ADH

Discussion Drawbacks

Incorrect preparation of trial 2 assay for 0.375M Ethanol Possible contamination when preparing NAD+

Inconsistent handling of cuvettes when loading into spectrophotometer Uniform mixing Time loading

Use less ADH enzyme to get a more accurate reading of the rate of reaction

Positive control – Lacking an inhibited version of the enzyme


Discussion Future Research

Investigate performance of ADH with other concentrations of ethanol under different temperatures or levels of pH

Run the reaction backwards by manipulating the equilibrium level

Use other versions of ADH and or types of alcohols Applications to medical industry involving alcoholism and

genetics


Discussion Application

Cirrhosis of liver – condition marked by chronic liver disease and subsequent degeneration


ReferencesBendinskas, K., DiJiacomo, C., Krill, A., & Vitz, E. 2005. Kinetics of

alcohol dehydrogenase – catalyzed oxidation of ethanol followed by visible spectroscopy, Journal of Chemical Education, 82(7), 1068-1070.

Edenberg, H. J. 2007. The genetics of alcohol metabolism: Role of alcohol dehydrogenase and aldehyde dehydrogenase variants. Alcohol Research & Health, 30(1), 5-13.

Voss, C. Gruber, K. Faber, T. Knaus, P. Macheroux, W. Kroutil. 2008. J. Am. Chem. Soc, 130, 13969-13972.

Laboratory Manual, 2012.Dr. Christenson


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Alcohol Dehydrogenase