Alcohol Dehydrogenase

Human Alcohol Dehydrogenase

VL & JP

Molecular Properties

Names

• Alcohol dehydrogenase class IV mu/sigma chain [6]

Active Site-ADH1

Molecular Characteristics- ADH1

• Gastric alcohol dehydrogenase [6]

• Retinol dehydrogenase [6]

• His-67, Cys-174, Cys-46, Thr-48, His-51, Ile-269, Val-292, Ala-317, and Leu-319

[9]

[13]

• Molecular Weight: 80 kDa [13]
• Optimal pH: 8.26-8.87 (Shore and Theorell 1966)
• Isoelectric Point: 6.23 [5]
• Extinction Coefficient: 189,320 cm-1 M-1

Regulation

[9]

[13]

[36]

Inhibition

• ADH susceptible to competitive inhibition with nitrogen heterocyclic compounds
• competes with ethanol to bind with zinc [38]

SREBP

• Other inhibitors: pyrazoles, heavy metals, thiourea, purine and pyrimidine derivatives [38]

• Activators: sulfhydryl activating reagents, mercaptoethanol, dithioreitol, cysteine [38]

Molecular Characteristics-ADH7

Enzyme Commission Number

Pyrazole Derivatives

• Sterol Regulatory Element-Binding Protein (SREBP) [7]
• SREBP-1 proteins negatively regulate rat Class I ADH expression [7]
• Ethanol suppresses SREBP-1 to produce disinhibition and consequent ADH induction [7]
• liver X receptor agonist T0901317 (a known activator of SREBP-1c transcription) [7]

• Inhibits by forming complex with ADH and NAD+ [32]

• Committee established in 1956 by the International Union of Biochemistry to standardize enzyme classification and nomenclature [10]

• Research has shown it can be used to treat methanol or ethylene glycol poisoning [32]

• Alcohol Dehydrogenase: EC 1.1.1.1 [1]

[33]

[16]

[26]

• NADH at 340 nm using an extinction coefficient of 6.22mM-1 cm-1

[7]

• PI: 8.12 [5]

Post-Transcriptional Modification

• ADH1 is regulated by transcription and/or post-transcriptional modification [7]

• Post-transcriptional modification of the mRNA has been found [7]

Enzyme Analysis

Alcohol Dehydrogenase Fun Facts

Essential

Compounds

Calculations

[21]

Alcoholism

• Sodium Phosphate Buffer pH ~8.8

[21]

• ADH1B*2, is common in people of Chinese, Japanese, and Korean descent [2]

[9]

• Divide by concentration to get specific activity

[14]

• Another version of the ADH enzyme, called ADH1B*3, occurs in 15 to 25 percent of African Americans. These enzymes metabolize alcohol to acetaldehyde very efficiently, leading to elevated acetaldehyde levels that make drinking unpleasant [2]

• Sodium Phosphate Buffer pH ~7.0

Hangovers

• ALDH1A1*2 and ALDH1A1*3, may be associated with alcoholism in African-American people [2]

ADH in Humans

• Acetaldehyde: highly reactive and toxic, can reach saturation point and escape into blood stream contributing to symptoms
• Breaking it down takes time! [34]

Purification

• Congeners: substances in alcoholic beverages includes alcetaldehyde, acetone, fuse oil, furfural, methanol, and others
• ADH metabolizes methanol into formaldehyde [35]

Forms and Functions

Why is it important?

• Produce recombinant protein (His-tag) in E.Coli

• Human bodies create at least nine different forms of ADH [19]

[39]

• Group of dehydrogenase enzymes that break down toxic alcohols to aldehye or ketone groups [19]

• Most are found in liver and lining of stomach [25]

• Lyse Cells

[39]

• Found in humans, animals, yeast, plants, and bacteria [19]

• In humans they also make important modifications to retinol, steroids, and fatty acids [37]

• Run through affinity chromatography
• Loading, washing, eluting

• Plays central role in alcoholic fermentation [37]

[39]

[18]

Biochemical pathways

Genetic Variations

• Humans have 7 different genes that encode ADH [25]

Mechanism

Primary Oxidative Pathway

• Found on long arm of chromosome 4 [25]

[20]

• Acetaldehyde: an intermediate in alcohol metabolism, is rapidly metabolized to acetate, is toxic to body [20]

• Acetate: oxidized to CO2, increases blood flow into liver and depresses central nervous system, also metabolized into acetyl CoA (lipid and cholesterol biosynthesis) [20]

[25]

[38]

• Oxidation of an alcohol to an adehyde in concert with transfer of a hydride to NAD [38]

• Differences in amino acids encoded by alleles can lead to differences in predicted rates of ethanol metabolism [25]

Consequences

• Hypoxia
• Ethanol metabolism increases hepatocytes' oxygen uptake from blood [20]

• Adduct formation
• Ethanol metabolism produces reactive molecules that can interact with amino acids [20]

• Changes in NADH/NAD+ levels [20]

• Tissue damage [20]

• Impairment of other metabolic processes [20]

• Cancer risk with chronic alcohol consumption [20]

ADH7 in Humans

References

Forms and Functions

• Amino acid sequence shows 59-70% identities with other human ADH classes [29]

• Plays regulatory role in biosynthetic pathway for retinoic acid [29]

Functions

[1] "ENZYME." Entry 1.1.1.1. ExPASy. Web. 10 Nov. 2014. <http://enzyme.expasy.org/EC/1.1.1.1>.

[2] "Alcohol Metabolism: An Update." NIAAA Publications. NIH. Web. 10 Nov. 2014. <http://pubs.niaaa.nih.gov/publications/AA72/AA72.htm> (Alcoholism)

[3] "RCSB Protein Data Bank - RCSB PDB - 1M6H Structure Summary." RCSB Protein Data Bank - RCSB PDB - 1M6H Structure Summary. Protein Data Bank. Web. 11 Nov. 2014. <http://www.rcsb.org/pdb/explore/explore.do?structureId=1m6h>. (Protein Image-3)

[4] "Section 16.4: Hydrogenation/dehydrogenation Reactions of Carbonyls, Imines, and Alcohols."Chemwiki. UC Davis. Web. 10 Nov. 2014. <http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_16:_Oxidation_and_reduction_reactions/Section_16.4:_Hydrogenation//dehydrogenation_reactions_of_carbonyls,_imines,_and_alcohols>. (Mechanism Image-2)

[5] "ADH7 Humans." Isoelectric Point Calculator. PhosphoSite Plus. Web. 10 Nov. 2014.<http://www.phosphosite.org/isoelectricCalcAction.do;jsessionid=195EB50CDFED9616D48EC023973E510B?id=20163&residues=6>.

[6] "Adh7 - Alcohol Dehydrogenase Class 4 Mu/sigma Chain - Mus Musculus (Mouse)."Adh7 - Alcohol Dehydrogenase Class 4 Mu/sigma Chain - Mus Musculus (Mouse). UniProt. Web. 10 Nov. 2014. <http://www.uniprot.org/uniprot/Q64437>.

[7] He, Ling. "Post-transcriptional Regulation of Sterol Regulatory Element-binding Protein-1 by Ethanol Induces Class I Alcohol Dehydrogenase in Rat Liver." Journal of Biological Chemistry (2004): 28113-8121. Print. (Suppression of ADH mRNA in FGC4 cells by T0901317 in the absence (but not presence) of 50 mm ethanol)

[8] Liu, Chang-Jun, Yuanheng Cai, Xuebin Zhang, Mingyue Gou, and Huijun Yang. "Tailoring Lignin Biosynthesis for Efficient and Sustainable Biofuel Production." Plant Biotechnology Journal: N/a. Print.

(Lignin synthesis)

[9] "Alcohol Dehydrogenase Assay."Worthington Biochemical Corporation. Worthington Biochemical Corporation. Web. 10 Nov. 2014. <http://www.worthington-biochem.com/adh/assay.html>.

[10] "Enzyme Commission." The Free Dictionary. Farlex, Inc., 1 Jan. 2009. Web. 11 Nov. 2014. <http://medical-dictionary.thefreedictionary.com/Enzyme Commission>.

[11] "RCSB Protein Data Bank - RCSB PDB - 1M6H Structure Summary." RCSB Protein Data Bank - RCSB PDB - 1M6H Structure Summary. Protein Data Bank. Web. 11 Nov. 2014. <http://www.rcsb.org/pdb/explore/explore.do?structureId=1m6h>.

[12] Larroy, Carol, Xavier Parés, and Josep A Biosca. "Characterization of a Saccharomyces Cerevisiae NADP(H)-dependent Alcohol Dehydrogenase (ADHVII), a Member of the Cinnamyl Alcohol Dehydrogenase Family." European Journal of Biochemistry 269 (2002): 5738-745. Print.

[13] "Alcohol Dehydrogenase (ADH) Enzymes."Alcohol Dehydrogenase ADH RSS. Turin University, 23 Apr. 2009. Web. 11 Nov. 2014. <http://flipper.diff.org/app/pathways/info/1948>.

• Uniquely expressed in esophagus and gastric mucosa [29]

[26]

Biochemical Pathway

• Influences blood alcohol levels and alcohol metabolism before its absorption into blood [29]

• Ethanol is not a good stubstrate unless at high concnetrations in digestive tract [31]

[27]

ADH-Other Organisms

• Contributes to first-pass metabolism [31]

• Inefficient in ethanol oxidation but most active as retinol dehydrogenase [28]

[27] Duester, Gregg. "Alcohol Dehydrogenase as a Critical Mediator of Retinoic Acid Synthesis from Vitamin A in the Mouse Embryo." The Journal of Nutrition 128.2 (1998): 459-62. Print.

[28] "Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine, 5 Oct. 2014. Web. 11 Nov. 2014. <http://www.ncbi.nlm.nih.gov/gene/131>.

[29] "ADH7 Gene." GeneCards. Weizmann Institute of Science, 1 Jan. 2014. Web. 11 Nov. 2014. <http://www.genecards.org/cgi-bin/carddisp.pl?gene=ADH7>.

[30] Birley, A. J., M. R. James, P. A. Dickson, G. W. Montgomery, A. C. Heath, J. B. Whitfield, and N. G. Martin. "Association of the Gastric Alcohol Dehydrogenase Gene ADH7 with Variation in Alcohol Metabolism."Human Molecular Genetics (2007): 179-89. Print.

[31] Yokoyama, H. "ADH7 - Alcohol Dehydrogenase 7 (class IV), Homo Sapiens." WikiGenes - Collaborative Publishing. US National Library of Medicine. Web. 11 Nov. 2014. <https://www.wikigenes.org/e/gene/e/131.html>

[32] Blomstrand, Rolf. "Pyrazoles as Inhibitors of Alcohol Oxidation and as Important Tools in Alcohol Research: An Approach to Therapy against Methanol Poisoning." Proceedings of the National Academy of Sciences 76.7 (1979): 3499-503. Print.

[33] Bernstein, Adam. "Five Unhealthy Habits That Can Harm Your Hearing." The Hearing Professionals. 1 Jan. 2011. Web. 11 Nov. 2014. <http://thehearingprofessionals.files.wordpress.com/2011/08>

[34] Roth, Klaus. "Chemistry of a Hangover - Alcohol and Its Consequences Part 3."ChemViews Magazine. Chemie in Unserer Zeit/Wiley-VCH, 6 July 2011. Web. 11 Nov. 2014. <http://www.chemistryviews.org/details/ezine/1080019/Chemistry_of_a_Hangover__Alcohol_and_its_Consequences_Part_3.html>.

[35] Minnick, Fred. "In Search of a Cure for the Dreaded Hangover." Scientific American Global RSS. Scientific American, Inc., 7 Mar. 2014. Web. 11 Nov. 2014. <http://www.scientificamerican.com/article/in-search-of-a-cure-for-the-dreaded-hangover/>.

[36] Personal photograph by author. 2001. By University of Wisconsin-Eau Claire.

[37] Goodsell, David. "Alcohol Dehydrogenase." RCSB PDB-101. 1 Jan. 2001. Web. 11 Nov. 2014.

[38] Harel, Michal, David Canner, Alexander Berchansky, and David Birrer. "Alcohol Dehydrogenase." 14 Aug. 2014. Web. 10 Nov. 2014. <http://www.proteopedia.org/wiki/index.php/A

[39] Willoughby, N.a., T. Kirschner, M.p. Smith, R. Hjorth, and N.j. Titchener-Hooker. "Immobilised Metal Ion Affinity Chromatography Purification of Alcohol Dehydrogenase from Baker’s Yeast Using an Expanded Bed Adsorption System." Journal of Chromatography A 840 (1999): 195-204. Print.

• Oxidation of retinol to retinaldehyde to produce retinoic acid [28]

• Retinoic acid: active metabolite of vitamin A, controls growth, development, and cell differentiation [28]

• Clearance of excess vitamin A to prevent toxicity [28]

Yeast

• alcohol + NADP+ <--> aldehyde + NADPH [22]

• Maintenance of proper NADP/NADPH [22]

Plants

[14] Ruiz, F. Xavier, Sergio Porté, Xavier Parés, and Jaume Farrés. "Biological Role of Aldo–Keto Reductases in Retinoic Acid Biosynthesis and Signaling." Frontiers in Pharmacology (2012). Print.

[15] Konturek, S.J., P.C. Konturek, and T. Pawlik. "DUODENAL MUCOSAL PROTECTION BY BICARBONATE SECRETION AND ITS MECHANISMS." (2004): 5-17. Print.

[16] "ADH7." Data Sheets. Santa Cruz Biotechnology, Inc. Web. 11 Nov. 2014. <http://datasheets.scbt.com/sc-161314.pdf>.

Konturek, S.J., P.C. Konturek, and T. Pawlik. "DUODENAL MUCOSAL PROTECTION BY BICARBONATE SECRETION AND ITS MECHANISMS." (2004): 5-17. Print.

[17] Xie, P. "X-ray Structure of Human Class IV Sigma Sigma Alcohol Dehydrogenase. STRUCTURAL BASIS FOR SUBSTRATE SPECIFICITY." Journal of

[18] Biological Chemistry272 (1997): 18558-8563. Journal of Biological Chemistry. Journal of Biological Chemistry. Web. 11 Nov. 2014. <http://www.jbc.org/content/272/30/18558.full.pdf>.

[19] Bullock, Clive. "The Biochemistry of Alcohol Metabolism — A Brief Review." Biochemical Education: 62-66. Print.

[20] "Overview: How Is Alcohol Metabolized by the Body?" NIAAA Publications. NIH. Web. 11 Nov. 2014. <http://pubs.niaaa.nih.gov/publications/arh294/245-255.htm>.

[21] "Enzymatic Assay of Alcohol Dehydrogenase (EC 1.1.1.1)." Sigma-Aldrich. Sigma-Aldrich, 1 Jan. 2014. Web. 11 Nov. 2014. <http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-alcohol-dehydrogenase.html>.

[22] Larroy C, et al. (2002) Characterization of a Saccharomyces cerevisiae NADP(H)-dependent alcohol dehydrogenase (ADHVII), a member of the cinnamyl alcohol dehydrogenase family. Eur J Biochem 269(22):5738-45

[23] Glazer, A. W., and Nikaido, H. (1995). Microbial Biotechnology: fundamentals of applied microbiology. San Francisco: W. H. Freeman, p. 340.

[24] Protein: Alcohol Dehydrogenase. The College of Saint Benedict and Saint John's University. 1 March 2010 <http://www.users.csbsju.edu/~hjakubow/classes/rasmolchime/99ch331proj/alcoholdehydro/index.htm>

[25] Edenbergh, Howard. "The Genetics of Alcohol Metabolism: Role of Alcohol Dehydrogenase and Aldehyde Dehydrogenase Variants." The Journal of the National Institute on Alcohol Abuse and Alcoholism 30.1 (2007): 5-13. Print.

[26] Logothetopoulos JH; Myant NB (1956). "Concentration of radioiodine and S-35-labeled thiocyanate by stomach of the hamster.". J Physiol 133: 213–219.

• ADH7 encodes for cinnamyl alcohol dehydrogases (CADs) [22]

• CADs are a subgroup of alcohol dehydrogenases that are involved in the biosynthesis of lignin (fills area in between cell walls, plays significant role in carbon cycle, useful in wood and paper products) [22]

[12]

Yeast

• Homodimer of 40 kDa subunits and a pI of 6.2-6.4 [22]

• Deletion of ADH7 was not lethal for the yeast [22]

• Could participate in the synthesis of fusel alcohols (isopropanol, methanol, aromatic alcohols), ligninolysis and NADP(H) homeostasis [22]