PRACTICAL-REPORT: Study of the enzyme Alcohol Dehydrogenase

ABSTRACT
Since the discovery of the enzyme Alcohol dehydrogenase, ADH, in the baker's yeast Saccharomyces cerevisiae in 1937, to the recent research on genetic studies of alcoholism, it has always suspected that the alcohol metabolism plays a critical role in the pharmacological and toxicological consequences arising therefrom. In addition, these enzymes also have numerous applications in the field of biotechnology, such as the biodegradation of aromatic hydrocarbons, the catalyzation of the fuel in fuel cells or the biotransformation of ethanol to synthesize enantiomerically pure stereoisomers of chiral alcohols. These are just some of the reasons why today are being carried out numerous studies on the structural and genetic basis of this enzyme. To this end, numerous strategies have been developed to facilitate the understanding of the biochemistry of this enzyme, and others, such as the different websites that allow the visualization of the 3D structure of proteins and enzymes, and of their active sites and cofactors. In our practice, we did a study of the Alcohol dehydrogenase using one of these web resources, the FirstGlance in Jmol.

KEYWORDS AND ABBREVIATIONS

ADH - Alcohol dehydrogenase 3D - tridimensional NAD - Nicotinamide Adenine Dinucleotide NADP - Nicotinamide Adenine Dinucleotide Phosphate MDRs - dehydrogenases/reductases medium chain SDRs - dehydrogenases/reductases short-chain NADH - reduced form of Nicotinamide Adenine Dinucleotide NADPH - reduced form of Nicotinamide Adenine Dinucleotide Phosphate S. cerevisiae - Saccharomyces cerevisiae H. sapiens - Homo sapiens

INTRODUCTION
The interconversion of alcohols, aldehydes and ketones are essential processes in both prokaryotic and eukaryotic organisms. The Alcohol dehydrogenase (ADH) is a dimeric enzyme with a molecular weight of 80 kDa, belonging to the class of oxidoreductases enzymes that are defined as enzymes that catalyze the reversible oxidation of alcohols to the corresponding aldehydes or ketones with the consequent reduction of NAD or NADP to NADH or NADPH. The reaction that catalyzes is: Alcohol + NAD+ aldehyde or ketone + NADH As cofactors in the reaction, zinc or iron can be used depending on the type of Alcohol dehydrogenase. There are five different classes of ADH that contain seven different human Alcohol dehydrogenases: Class I: that consists in three different subunits: , , and that are encoded by the genes ADH1A, ADH1B, and ADH1C, so there are 3 different ADH1 and are the following: Alcohol dehydrogenase 1A (ADH1A) or Alcohol dehydrogenase alpha subunit. Alcohol dehydrogenase 1B (ADH1B) or Alcohol dehydrogenase beta subunit. There are three known alleles for this enzyme, ADH1B*1 variant for beta-1, ADH1B*2 variant for beta-2 and ADH1B*3 variant for beta-3. The allele frequency of ADH1B*2 is approximately 75% in the eastern population while is less than a 5% in the caucasian population. Alcohol dehydrogenase 1C (ADH1C) or Alcohol dehydrogenase gamma subunit. There are two known major alleles for this enzyme, the ADH3*1 or gamma-1 that has Arg-272/Ile-350 and the ADH3*2 or gamma-2 that has Gln-273/Val-350.

The ADH 1 is found in the cytoplasm and has 2 atoms of zinc bounded to each subunit. Each subunit consists of a dimer with units 1A, 1B and 1C identical (homodimer) or non-identical (heterodimer). Class II: Alcohol dehydrogenase 4 (ADH4) or polypeptide. It is located in the cytoplasm and has bounded two zinc atoms per subunit, which is a homodimer.

Class III: Alcohol dehydrogenase 5 (ADH5). This enzyme is remarkably ineffective in the oxidation of ethanol, but correctly catalyzes the oxidation of primary alcohols of long-chain and the oxidation of S-hydroxymethyl glutathione. It is located in the cytoplasm and has coupled two zinc atoms per subunit, which is a homodimer. Class IV: Alcohol dehydrogenase 7 (ADH7) or / polypeptide. This enzyme participates in the oxidation of retinol for the retinoic acid synthesis, an important hormone for the cellular differentiation. Medium-chain compounds (octanol) and aromatic (m-nitrobenzaldehyde) compounds are the best substrates. The ethanol is not a good substrate but when this reaches high concentrations in the digestive tract, the enzyme participates in ethanol oxidation and contributes to the first step of the metabolism of ethanol. This enzyme is found in the cytoplasm of stomach cells and binds two zinc atoms per subunit, which is a homodimer. ClassV: Alcohol dehydrogenase 6 (ADH6). It is located in the cytoplasm and has bounded two atoms of zinc per subunit. This enzyme is more specific in stomach and the liver tissues. The ADH allows the organism to alcohol consumption, but its evolutionary purpose is probably breaking alcohols that are contained in the food in a natural way, or that are produced by bacteria in the digestive tract. Another possibility is that the evolutionary purpose of the enzyme is the metabolism of the vitamin A. In our practice we analyze the structure of the human Class I ADH and the yeast ADH, and we compare the structural differences that they present, making use of the software available to perform this task.

METHODS
Part 1: Alcohol Dehydrogenase Reaction Using the database of enzymes Brenda, we explore the following properties of the ADH of Homo sapiens: Molecular Weight: 39500-isozyme ADH3, apparent molecular weight deduced from electrophoretic mobility 39720-isozyme ADH3, calculated from amino acid sequence 39870-isozyme ADH1C, calculated from amino acid sequence 40000-isozyme ADH2, apparent molecular weight deduced from electrophoretic mobility; isozyme ADH4, calculated from amino acid sequence Subunits number: Dimer-2 * 40000, SDS-PAGE Dimer-2 * 40000, SDS-PAGE; 2 * 41000, class III isoenzyme chi ADH, SDS-PAGE Dimer-2 * 42000, anodic enzyme form, SDS-PAGE Also we compared the different substrates / products of this enzyme for Homo sapiens and S. cerevisiae: Saccharomyces cerevisiae SUBSTRATE (R)-2-butanol + NAD+ (R,S)-2-methylbutan-1-ol + NAD+ (S)-2-butanol + NAD+ (S)-2-butanol + NAD+ (S)-2-methylbutan-1-ol + NAD+ 2-propanol + NAD(P)+ 2-propanol + NAD+ 2-propanol + NAD+ PRODUCT (R)-2-butanone + NADH + H+ (R,S)-2-methyl-butan-1-one + NADH + H+ (S)-2-butanone + NADH + H+ 2-butanone + NADH (S)-2-methyl-butan-1-one + NADH + H+ acetone + NAD(P)H acetone + NADH acetone + NADH + H+

3-methylbutan-1-ol + NAD+ 2-methylpropan-1-ol + NAD+

3-methyl-butan-1-one + NADH + H+ 2-methyl-propan-1-one + NADH + H+

Homo sapiens SUBSTRATE 1-hydroxymethyl-6-methylpyrene + NAD+ 1-hydroxymethyl-8-methylpyrene + NAD+ 1-hydroxymethylpyrene + NAD+ 1-octanol + NAD+ 11-cis-retinol + NAD+ 12-hydroxydodecanoate + NAD+ 16-hydroxyhexadecanoate + NAD+ 17beta-hydroxyetiocholan-3-one + NAD+ 2-deoxy-D-ribose + NAD+ 2-hydroxymethylpyrene + NAD+ 3-phenyl-1-propanol + NAD+ PRODUCT 1-formyl-6-methylpyrene + NADH + H+ 1-formyl-8-methylpyrene + NADH + H+ 1-formylpyrene + NADH + H+ octanal + NADH + H+ 11-cis-retinal + NADH 12-oxododecanoic acid + NADH 16-oxohexadecanoic acid + NADH ethiocholan-3,17-dione + NADH ? + NADH 2-formylpyrene + NADH + H+ 3-phenyl-1-propanone + NADH

Part 2: Analysis of primary structure of ADH Using the software available on www.expasy.ch, we obtained physico-chemical information from the amino acid sequence of the following enzymes: ADH1 (P07327)

Theoretical isoelectric point (pI): 8.26 Molecular weight (Mw): 39858.69 (Da) ADH2 (P00325)

Theoretical isoelectric point (pI): 8.87 Molecular weight (Mw): 48217.56 (Da)

Part 3: Prediction of the secondary structure of a protein In this part of the practice, using one of the software tools we predicted the secondary structure of the enzyme from its amino acid sequence. Part 4: Comparison of the predicted structure with the actual structure Now we compare the structure that we obtained above with the actual structure of the protein, which we can obtain from the database of protein structures housed in the http://www.rcsb.org page. On this page the actual structure of the protein is named 1HSO. Part 5: Identification of residues of catalytic/structural importance Finally, in this part of the practice, we study the 3D structure of the enzyme to identify the catalytic and structural residues with importance. This was made possible by a molecular viewer hosted on the website of Proteopedia

(http://www.proteopedia.org/wiki/index.php/Main_Page), the FirstGlance in Jmol. Thus we identified the ADH ligands, which were the following: NAD: Nicotinamide Adenine Dinucleotide PYZ: 4-Iodopyrazole ZN: Zinc Ion

And also the ADH residues that interact with these ligands: NAD: Thr 178(A), Gly 201(A), Val 268(A), Gly 293(A), Ile 318(A), Cys 46(A), Cys 174(A), Leu 200(A), Leu 362(A), Gly 199(A), Asn 225(A), Arg 271(A) and Leu 309(B). PYZ: Met 57(A), Val 116(A), Ile 318(A), Val 294(A) and Met 306(B). ZN1: Cys 111(A), Cys 97(A), Cys 103(A) and Cys 100(A). ZN2: Asp 49(A), Cys 174(A), His 67(A) and Cys 46(A).

In addition we identified the amino acid residues that are associated with domains 1 and 2 of each subunit of the enzyme: Domain 1: 1-178 and 318-374 Domain 2: 179-317

RESULTS
Part 1: Comparison of the different substrates / products of this enzyme for Homo sapiens and S. cerevisiae: In relation to the differences between the different substrates of the enzyme of H. sapiens and of S. cerevisiae we observed that the substrates of human ADH are both simple structure alcohols and alcohols of structure more complex, such as secondary cyclic alcohols. In contrast, the substrates of the yeast ADH are alcohols with more simple structure, ie, primary, secondary or hemiacetals alcohols, but never secondary cyclic alcohols. S. cerevisiae Homo sapiens

2-methylpropan-1ol

3-phenyl-1-propanol

Part 2: Predict the location of this protein on 2D gel encompassing the pH range of pH 7 to pH 10 and molecular weight range of 10kDa to 100kDa. 7 100 -90 -80 -Mw (KDa) 70 -60 -50 -40 -20 -10 -ADH1 ADH2

pI

10

Parts 3 and 4: Prediction of the secondary structure of the protein and comparison with the real structure. In general, the predicted structure for the enzyme by the program resembles quite well with the actual structure of the enzyme, only small differences are observed in some regions, because of the exact position of the different alpha or beta helices.

Real Structure:

Part 5: Identification of residues of catalytic/structural importance

NAD:

PYZ:

 ZN1:

ZN2:

DISCUSSION
In our results we see that there are significant differences in the structure and therefore in the substrates for those that the enzyme can catalyze the oxidation-reduction reaction, depending if the enzyme belongs to humans or to yeast. Without the help of programs like the one we used it would be very difficult to analyze the differences between the enzymes and proteins existing. The advancement of the new technologies has promoted the study of biochemistry and proteomics, two very important sciences today, because of the repercussion their discoveries have in the health and in the environmental.

CONCLUSION
Nowadays thanks to technological advances, we can get in a few minutes or hours the results that before took weeks to get. For this reason it is important that those who move in the field of science, have some basic knowledge on how to handle the tools available. Studies in relation to the enzyme ADH are on the order of day due to its role in environmental conservation and due also to its role as the principal catalyst for the conversion reaction of alcohol in acetaldehyde, and to the existing isoforms of this enzyme that has implications in the differences of the alcohol metabolism in different individuals. Its being currently investigated the involvement of different genetic polymorphisms in candidate genes related to ethanol metabolism with a possible genetic susceptibility to alcohol consumption. Hence that the development of advanced techniques, that allow us to analyze these and other polymorphisms have so much importance to our society.

REFERENCES
Gloria Anglica Gonzlez Hernndez, Juan Carlos Torres Guzmn, J. Flix Gutirrez Corona y Roberto Zazueta Sandoval, Alcohol Deshidrogenasas Fngicas: Papel Fisiolgico y Potencial Biotecnolgico, Revista Enlace Qumico, nmero 17. http://revistaequim.com/numeros/17/deshidrogenasa.htm David L. Nelson, Michael M. Cox, Lehninger, Principios de Bioqumica, 4 edicin, Editorial Omega. (2005). Donald Voet, Judith G. Voet, Pratt, Fundamentos De Bioqumica, 2 edicin, Editorial Mdica Panamericana. (2007). Agarwal, D.P., Molecular genetic aspects of alcohol metabolism and alcoholism, Pharmacopsych., 30: 79-84 (1997). http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1hso http://www.proteopedia.org/wiki/index.php/1hso http://www.rcsb.org/pdb/explore/explore.do?structureId=1HSO https://www.predictprotein.org/get_results.php?req_id=285270 Predisposicin gentica en el consumo de alcohol: el caso de la Alcohol Deshidrogenasa 1C._ http://scielo.isciii.es/pdf/cmf/n48-49/art04.pdf