mALDH and Dehydrogenation of Aromatic Aldehydes
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 275
forces, relate to perturbations in the distribution of
electrons between atoms. In contrast, hydrophobic in-
teractions have been largely explained in terms of London
forces, which are the weakest of the three types of van
der Waals interactions. Because the present study
described the correlation of log Km with 0ø rather than
with hydrophobic character (e.g., log P), it is likely that
substrate behavior is dependent upon van der Waals
interactions other than London forces.
Effective molecular volume is greater where there is
hydrogen bonding to substituent groups. Koehler et al.,
have defined the isotropic surface, which is essentially
the surface area of the nonpolar region of a molecule that
is accessible to nonspecific interactions (32). Hydrophilic
regions, such as hydroxyl or carboxyl substituents with
their associated waters of solvation, are excluded from
the calculation of the isotropic surface. Thus, interaction
of an enzyme and substrate would be a balance between
favorable (attractive) van der Waals interactions, be-
tween amino acid residues at the active center and the
isotropic surface of the molecule, and unfavorable (re-
pulsive) interactions due to steric and electronic factors.
In the case of the mALDH, the presence of hydrophilic
substituents in the 3- or 4-positions of benzaldehyde may
sterically hinder the appropriate orientation of the
substrate at the active center. Similarly the low activity
of benzaldehydes possessing large substituents in both
the 3- and 4-positions may be due to steric hindrance. In
such cases, unfavorable interactions outweigh favorable
ones.
The possible orientation of 3-phenoxybenzaldehyde in
the active site of the mALDH is shown in Figure 5. The
approximate dimensions of this molecule would be 7.5 Å
by 12 Å, including the van der Waals radii, and the
aromatic system itself, including the π orbitals, is be-
tween 5 and 6 Å deep. This structure and the observed
rates of substrate dehydrogenation could be accom-
modated if the active site of the mALDH contains a
relatively narrow cleft (as depicted in Figure 5). This
model also accounts for the unfavorable effect of ortho
substitution on substrate behavior. Adverse steric in-
teractions between an ortho substituent and either the
cofactor (NAD, bound at the adjacent cofactor site) or the
substrate cleft itself are probably responsible for the
undetectable rates of dehydrogenation of these sub-
strates.
nucleophilic attack of the Cys-302 sulfur at the carbonyl
carbon of the substrate to form a thiohemiacetal, followed
by hydride transfer from the substrate to NAD and the
formation of a thioester intermediate (5). Although yet
to be demonstrated directly for each ALDH, sequence
alignments suggest that Cys-241 is probably the corre-
sponding residue in the mALDH (5, 10). Hydrolysis of
the thioester releases the carboxylic acid product and
regenerates the enzyme, which can accept further sub-
strate molecules. The presence of electron-withdrawing
substituents (σ positive) would be expected to favor
dehydrogenation by enhancing the partial positive char-
acter of the carbonyl carbon. This was noted in the
present study as well as in another study of benzalde-
hydes containing simple substituents as substrates for
a bacterial ALDH (33).
The functional role of the mALDH has yet to be
completely clarified. It has been suggested that the
enzyme may detoxify aldehydic products of microsomal
lipid peroxidation (12). Such toxic aldehydes, including
alkanals, alkenals, ketones, and hydroxyalkenals, are
generated in vitro during lipid peroxidation in carbon
tetrachloride- or iron-ADP-containing systems (34). It
is feasible that the enzyme may function in the elimina-
tion of alkanals and alkenals since the quantities of these
agents that are formed during lipid peroxidation would
be adequate for saturation of the mALDH (>Km). From
the present analysis, ALDHs may also have a role in the
dehydrogenation of xenobiotic aromatic aldehydes, and
those formed in vivo from oxidation of neurotransmitters,
to the corresponding aryloxybenzoic acids.
Ack n ow led gm en t. This study was supported by a
grant from the Australian National Health and Medical
Research Council. R.M. was the recipient of an Astra/
Gastroenterological Society of Australia Career Develop-
ment Award.
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The model of the mALDH active site in Figure 5 takes
into account the apparent importance of the shapes of
the 3- and 4-substituents as determinants of Km. The
zero-order shape attribute of the 4-substituent in the
aromatic aldehydes (0κ4) has been likened to the property
of symmetry, whereas the third-order shape attribute of
the 3-substituent (3κ3) is believed to encode a feature of
central branching (24). The final parameter that ap-
peared in the best regression equation was the Hammett
constant of the 4-substituent (σ4). Aldehydes containing
electron-donating groups, such as methyl and methoxy,
were substrates inferior to benzaldehyde. Strongly elec-
tron-withdrawing groups in the 4-position, such as nitro
and cyano, promoted dehydrogenation. The complex
nature of the 3-substituent in several of the substrates
led to problems in the detailed assessment of any
electronic effect at this position. However, the electronic
effects of substituents of the phenoxy system should be
insignificant at the benzaldehyde carbonyl group.
The catalytic mechanism of the mitochondrial ALDH
is depicted in Scheme 1 and appears to involve the initial