Amity Institute Of Biotechnology
Amity Institute Of Biotechnology
M.Sc(BT)
Enzyme and Enzyme Technology(GCM 718)
Dr. Benu Kumar
Amity Institute Of Biotechnology
Alcohol Dehydrogenase(ADH)-Introduction
• Alcohol Dehydrogenase belongs to the oxidoreductase family of enzymes
catalyzes the interconversion between alcohols and aldehydes or ketones with
the reduction of NAD+ or NADP+ to NADH or NADPH
ADH
Ethanol + NAD+ ---------→ Acetaldehyde + NADH
• Molecular weight of enzyme is 146.8KDa, found in animals, plant, fungi, algae,
bacteria and other related species.ADH is found in high concentrations within
the human liver and kidney
• Enzyme is composed of 347 amino acids and its isoelectric point is 6.23
• Humans have at least nine known forms of ADH
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Structure Of Alcohol Dehydrogenase
Horse liver alcohol dehydrogenase is a dimer
Each monomer has 2 subunits- a binding site for one NAD+ and two Zn2+:although only
one zinc ions is directly involved in catalysis
His-105 and Tyr-286 on each monomer interact with each other to seal the packing
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Active Site Characteristics
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
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Binding and Interaction at Active Site of ADH
NAD+ binds at residues 293-298
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 protein go from its
apo "open" form to the halo "closed". This
narrows the cleft, brings the substrate
The hydrophobic pocket:- Leu-57, Phe-93,
binding site closer and excludes water Leu-116, Phe-110, Phe-140, Leu-141, Val-294,
from the active site which is vital for the Pro-295 and Ile-318 (red). Zinc (orange), Cys-
174 (purple), Cys-46 (yellow) and His-67
activity of ADH (green) Cxf (in this case) in blue and oxygen
involved in the dehydrogenation reaction
shown in white
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Reaction Mechanism:
The initial proton loss from the substrate in the hydrophobic environment of the active
site to the surface of the enzyme appears to involve histidine-5I, the 2 'hydroxyl group
of the coenzyme and serine-48, possibly operating in a coordinated fashion.
The zinc ion then acts as an electrostatic catalyst to stabilize the negatively
charged transition-state, while a hydride ion is transferred to NAD+, becoming the HR
atom of NADH.
For the reverse reaction, the zinc ion acts as an electrophilic catalyst to enhance the
polarization of the carbonyl group of the substrate and facilitate hydride transfer from
the reduced coenzyme.
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Summary
• Binding of the ADH enzyme to the coenzyme NAD+ for functional
activation.
• Binding of the alcohol (as substrate) with ADH.
• This binding of enzyme and substrate is facilitated with coordination to
zinc atom.
• Deprotonation in the enzyme at Histidine 51 position.
• This deprotonation is subsequently followed by deprotonation of
nicotinamide ribose.
• Deprotonation of another residue in protein, Threonine 48, for
accompanying the alcohol molecule.
• Final deprotonation of the alcohol molecule
• Formation and release of the final product (aldehyde).
