Alcohol Dehydrogenase The Hang-Over Enzyme Pat Baron

Outline • Why the Hang-Over enzyme? • Forms, Functions, and a little Fiction • A closer look into the active site of ADH • Conclusions

Why the Hang-Over Enzyme? - General Information on ADH and questions answered! • Alcohol Dehydrogenase belongs to the oxidoreductase family of enzymes • ADH is found in high concentrations within the human liver and kidney • The primary and most common role of ADH in humans is to detoxify incoming ethanol by converting it into acetaldehyde • The resulting acetaldehyde, a more toxic molecule than ethanol, is quickly converted into acetate and other molecules easily utilized by the cell

But Why the Hang-Over? - Partially explained by the chemistry • Incoming ethanol is converted to acetaldehyde by the mechanism below: Ethanol + NAD+ ----- Acetaldehyde + NADH • Ethanol is oxidized by ADH into acetaldehyde and NADH is formed • The highly toxic acetaldehyde is then further modified into foodstuffs for the cell • Throughout these processes, water molecules are lost and dehydration could set in after prolonged imbibing.

There you have it! Dehydration is the Culprit • “…. then dying of thirst must be one of the worst hang-overs to experience. ” A Beautiful Mind

Forms, Functions, and a little fiction - structure and function of ADH and associated isoenzymes • Humans have at least nine known forms of ADH • ADH exists as a homo or heterodimer due to the fact there are two different types of monomer • The two types are E and S for ethanol active and steroid active respectively. Although they have different specificities, both are nearly identical at 374 aa’s long • Therefore, possible types of ADH are: EE, SS, and ES hybrid ADH. • EE is the most commonly found at 40 -60%

Characteristics of EE ADH • EE ADH has a molecular weight of about 80 000 • There are 8 chains, 60 helices, and 74 beta strands in ADH • Each monomer of the dimer has 2 subunits • Each of the two subunits has a binding site for one NAD+ and two Zn 2+ (seen later) • Activated by cyanate (NCO) and inhibited by heavy metals and chelating agents

For the Microbiologist in all of us • Three distinct genes are responsible for the production of ADH • However, gene products show a 93% homology • Cross-species homology exists as well

Homology Between Species • Human EE ADH • Equine EE ADH

Interaction of Monomers • Two residues are directly responsible for the monomer packing of ADH • His-105 and Tyr-286 on each monomer interact with each other to seal the packing • The ring side-chains of His-105 will stack on top of the Tyr-286 side chain on the other monomer • The monomers are aligned anti-paralell to each other

Functions in Industry • Alcohol fermentation is an industrial process to make alcoholic beverages, and directly involves ADH isolated from yeast • Specifically, this is the conversion of glucose to ethanol as seen below: Glucose + 2 Pi + 2 ADP +2 H+----- 2 Ethanol +2 CO 2 + 2 ATP +2 H 20 • The above reaction is catalyzed by yeast ADH

• Yeast ADH is much larger than Human/Equine

A Little Fiction • Hypothetical: Life without EE ADH • Assumptions: All normal functions of EE ADH are still working properly, however, the oxidation of ethanol mechanism is faulty… • Result?

The Homer Hypothesis • If ethanol could not be converted to acetaldehyde, any alcohol that is ingested would remain in its “toxic” form • Constant state of inebriation?

A Closer Look into the Active Site of ADH -an in depth look at the interactions taking place in the heart of ADH

Active Site Characteristics of ADH • As mentioned earlier, each subunit of one monomer contains one binding site for NAD+ and two binding sites for Zn 2+ • Each Zinc ion is ligated directly between the side chains of Cys-46, His-67, Cys-174 and a water molecule which is hydrogen bonded to Ser-48. • Between the two binding sites where the zinc is located, there are two clefts. One which binds NAD+ and one which binds the substrate (ethanol)

• Zinc bound to Cys-46, His-67, Cys-174, and Ser-48 (Blue) and the coenzyme NAD+ (purple) attached to His-51 (yellow) and Lys-228 (cyan). The eight zinc molecules are in red. The four zincs seen easily are not directly involved in the proton transfer chain.

Components and Interactions at the Binding Site of ADH • NAD+ is the coenzyme for ADH and is absolutely necessary for the conversion of ethanol • One molecule of NAD+ is used to convert ethanol to acetaldehyde by proton transfer • During hydrogen transfer, two hydrogens are stripped off the ethanol by zinc

Conformation Change at the Active Site • NAD+ binds at residues 293 -298 and causes a 100 rotation • This causes the catalytic domain to move closer to the coenzyme binding domain and closes the active site cleft • S 48 helps in the proton relay system

But I Must Know More! • The two active sites are in clefts between the coenzyme binding core and the catalytic domains • Ethanol binds to the hydrophobic core lined by nine amino acids, which surround the substrate • After binding NAD+, the 100 rotation makes the protein go from its apo "open" form to the halo "closed". This narrows the cleft, brings the substrate binding site closer and excludes water from the active site which is vital for the activity of ADH

• The hydrophobic pocket: - Leu-57, Phe-93, Leu-116, Phe-110, Phe -140, Leu-141, Val-294, Pro-295 and Ile-318 (red). Zinc (orange), Cys-174 (purple), Cys-46 (yellow) and His-67 (green) Cxf (in this case) in blue and oxygen involved in the dehydrogenation reaction shown in white

A Closer Look • The zinc atom is held in place by cysteine 46 to the left, cysteine 174 to the right, and histidine 67 above. Ethanol binds to the zinc, and the NAD analog extends below the ethanol

Conclusions • Alcohol Dehydrogenase is the Human Body’s offensive line (colts) against alcoholic toxins being ingested • ADH substrate specificity is broad, with most alcohols being potential targets (eg. Methanol Formaldehyde) • Once bound to zinc, however, a conformation change ensures tight binding. • Homer Hypothesis is not feasible

References: • • • 1. Adolph HW, Zwart P, Meijers R, Hubatsch I, Kiefer M, Lamzin V, Cedergren-Zeppezauer E. (2000). Structural basis for substrate specificity differences of horse liver alcohol dehydrogenase isozymes. Biochem 30 (42), 12885 -97 2. Niefind K, Riebel B, Muller J, Hummel W, Schomburg D. (2000). Crystallization and preliminary characterization of crystals of Ralcohol dehydrogenase from Lactobacillus brevis. Acta Crystallogr D Biol Crystallogr 56, 1696 -8 3. Hawkins SW, Boudet AM. (1994). Purification and Characterization of Cinnamyl Alcohol Dehydrogenase Isoforms from the Periderm of Eucalyptus gunnii Hook. Plant Physiol 104 (1), 75 -84 4. http: //bssv 01. lancs. ac. uk/Stu. Work/BIOS 31601/adh/horse%20 liver 5. http: //www. rcsb. org/pdb/molecules/pdb 13_1. html 6. http: //www. worthington-biochem. com/manual/A/ADH. html