Transcript Alcohol Dehydrogenase - University of Guelph

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 Zn2+ (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 + 2Pi + 2ADP +2H+----- 2 Ethanol +2CO2 + 2ATP +2H20
• 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 Zn2+
• 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
• S48 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:
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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://bssv01.lancs.ac.uk/StuWork/BIOS316/BIOS31601/adh/horse%20liver
5. http://www.rcsb.org/pdb/molecules/pdb13_1.html
6. http://www.worthington-biochem.com/manual/A/ADH.html