Human alcohol dehydrogenase (ADH):

Structural-functional relationships
JAN ROMMEL DUTERTE
BS Chemistry – 3
University of San Carlos, Nasipit, Talamban, Cebu City 6000
Abstract Alcohol dehydrogenase is an important enzyme in the human body that detoxifies ingested
alcohol. Its structure is discussed and related to its functions in the body.

Alcohol dehydrogenase (ADH) is a zinc metalloenzyme responsible for the oxidation of alcohols to
aldehydes by reaction with NAD+, and the reduction of aldehydes to alcohols by NADH (Brändén et
al., 1975). In mammals, the ADH system is divided into six classes, ADH1-ADH6, five of which are
present in humans (Jörnvall et al., 1999). Five structural genes in humans, ADH1 to ADH5, encode for
five different polypeptide subunits—α, β, γ, π and χ, respectively. The α, β, and γ subunits can
combine randomly to form homodimeric (αα, ββ, γγ) or heterodimeric (e.g. αβ, αγ) isoenzymes; these
are called ADH1 class.
Several researchers have reported on ADH’s wide specificity. It catalyzes the oxidation of several
primary, secondary and cyclic alcohols, as well as the reduction of many aldehydes and ketones (Sund
& Theorell, 1963). ADH is our body’s line of defense against a particularly common toxin in the
environment: it catalyzes the reaction between NAD+ and ethanol to form NADH and acetaldehyde,
which then further reacts with NAD+ to form harmless acetic acid.

Figure 1. Ethanol metabolism in humans

ADH is composed of two protein sub-units of 40,000 daltons

Figure 2. Active site of ADH
(Brändén et al., 1975). The nicotinamide of the NAD is bound near the zinc atom by one of the two
protein sub-units.
each. One of the sub-units is a catalytic domain while the other
is a NAD-binding domain, and both NAD and substrate bind in
a pocket between the two sub-units (Svensson et al., 2000).
The pocket contains the catalytic zinc atom of the enzyme,
coordinated to three amino acids: Cys 46, Cys 174 and His 67
(left, right and above the zinc atom in Figure 2); the alcohol’s
hydroxyl ion (or a water molecule) completes the tetragonal
coordination by projecting into the active site pocket
 Aldehyde formation during the catalytic action of the enzyme requires a net removal of two
hydrogens from the alcohol substrate. This dehydrogenation process is known to proceed by a
mechanism of combined proton and hydride ion transfer, and, as shown in Figure 3, the hydride
transfer occurs directly from substrate to coenzyme (Brändén et al., 1975).
Kinetic studies on ADH have revealed the series of
steps during the conversion of alcohols into
aldehydes (Figure 3). First, the coenzyme NAD+
binds to the enzyme; at the same time, the alcohol
coordinates with the zinc atom in the active site. The
alcohol is then deprotonated by a proton transfer
relay between Ser 48, His 51 and the NAD molecule;
Crystal structures indicate that His 51 deprotonates
the nicotinamide ribose, which deprotonates Ser 48
and which eventually deprotonates the alcohol.
Hydride transfer from the resulting alkoxide
protonates NAD+, leading to NADH and a zinc-bound
aldehyde. The resulting aldehyde is then released,
and NADH dissociates from the enzyme (Hammes- Figure 3. Schematic illustration of the ADH active site
with a benzyl alcohol substrate and NAD+ cofactor
Schiffer & Benkovic, 2006).
Experiments done by Dalziel and Dickinson (1966) confirm that aside from primary alcohols,
secondary alcohols are also substrates for ADH. Their experiments show that secondary alcohols,
such as 2-propanol and 2-butanol, are poorer substrates than their isomeric primary alcohols as
indicated by their larger Michaelis constants. It can be argued that one reason for this lies in the
structure of the alcohol and the enzyme: the active site is a deep pocket, and it is much easier for a
primary alcohol to protrude into it than a secondary one. Even among primary alcohols, Dalziel and
Dickinson showed that branching also affects the strength of affinity since the branched-chain 2-
methyl-1-propanol is a poorer substrate (has a higher Michaelis constant) than straight-chain 1-
propanol and; they concluded that the Michaelis constant for the ADH substrate decreases with
increasing chain length.
Further experiments even show a difference in substrate affinity between different ADH classes.
Table 1. Kinetic constants for human and rat ADHs For example, while all ADH
classes can participate in ethanol
oxidation, ADH3 is not able to
saturate with ethanol and ADH2
shows only traces of ethanol
dehydrogenase activity. Of the
three homodimeric ADH1 forms,
the ββ form strongly binds to
alcohol while the αα form binds
the least; however, the γγ form is
the most efficient because it has
the highest turnover number,
Kcat, of the three homodimeric forms. ADH2 has a significantly higher Km than the ADH1 isoforms and

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therefore only makes a significant contribution to ethanol metabolism at high concentrations (Höög
et al., 2001).
It is interesting to note that changes in amino acid sequences can change the activity of ADH. Human
ADH2, for example, has an arginine at position 47 and histidine at 51; rat ADH2, on the other hand,
has a proline at 47 and threonine at 51. Rat ADH2, as presented in table 1, has very little ethanol
metabolizing activity compared to human ADH2. This is because of the proton transfer relay that
allows for the deprotonation of the alcohol substrate: for this to be possible, there has to be a histidine
residue at either position 47 or position 51 By replacing the rat’s proline with histidine at position
47, ADH activity can be restored (Höög et al., 2001).
CONCLUSIONS
Alcohol dehydrogenase is our primary defense against alcohol, a toxic molecule that compromises
the function of our nervous system. The high levels of alcohol dehydrogenase in our liver and stomach
detoxify about one stiff drink each hour. The alcohol is converted to acetaldehyde, an even more toxic
molecule, which is then quickly converted into acetate and other molecules that are easily utilized by
our cells. Thus, a potentially dangerous molecule is converted, through alcohol dehydrogenase, into
a mere foodstuff. Alcohol dehydrogenase also modifies other alcohols, often producing dangerous
products. For instance, methanol, which is commonly used to "denature" ethanol rendering it
undrinkable, is converted into formaldehyde by alcohol dehydrogenase. The formaldehyde then does
the damage, attacking proteins and embalming them. Small amounts of methanol cause blindness, as
the sensitive proteins in the retina are attacked, and larger amounts, perhaps a glassful, lead to
widespread damage and death.
Amino acid sequences have large effects on the activity of alcohol dehydrogenase. The proton
transfer relay that is the heart of the enzyme’s function is dependent on the presence of a single
histidine residue, and this speaks volumes about the relationship between the structure and the
specificity and function of an enzyme.

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References
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