New insights into the half-of-the-sites reactivity of human aldehyde dehydrogenase 1A1
Por:
Yoval-Sánchez B., Pardo J.P., Rodríguez-Zavala J.S.
Publicada:
1 ago 2013
Resumen:
Aldehyde dehydrogenases (ALDHs) couple the oxidation of aldehydes to the
reduction of NAD(P)(+). These enzymes have gained importance as they
have been related to the detoxification of aldehydes generated in
several diseases involving oxidative stress. It has been determined that
tetrameric ALDHs work only with two of their four active sites
(half-of-the-sites reactivity), but the mechanistic reason for this
feature remains unknown. In this study, tetrameric human aldehyde
dehydrogenase class 1A1 (ALDH1A1) was dimerized to study the correlation
of the oligomeric structure with the presence of half-of-the-sites
reactivity. Stable dimers from ALDH1A1 were generated by combining the
mutation of two residues of the dimer-dimer interface in the tetramer
(previously shown to render a low-active and unstable enzyme) and the
fusion of green fluorescent protein (GFP) in the C-terminus of the
mutant. Some kinetic properties of the GFP-fusion mutant resembled those
of human aldehyde dehydrogenase class 3A1, a native dimer, in that the
fusion dimer did not show burst in the generation of nicotinamide
adenine dinucleotide (NADH) and was less sensitive to the action of
specific modulators. The presence of primary isotope effect indicated
that the rate-limiting step changed from NADH release to hydride
transfer. The mutant showed higher activity with malondialdehyde and
acrolein and was more resistant to inactivation by acrolein compared
with the wild type. The mutant kinetic profile showed two hyperbolic
components when the substrates were varied, suggesting the presence of
two active sites with different affinities and catalytic capacities. In
conclusion, the ALDH1A1-GFP dimeric mutant exhibits full site
reactivity, suggesting that only the tetrameric structure induces the
half-of-the-sites reactivity. Proteins 2013; 81:1330-1339. (c) 2013
Wiley Periodicals, Inc.
Filiaciones:
Yoval-Sánchez B.:
Departamento de Bioquímica, Instituto Nacional de Cardiología, México D.F., Mexico
Pardo J.P.:
Univ Nacl Autonoma Mexico, Fac Med, Dept Bioquim, Mexico City 04510, DF, Mexico
Rodríguez-Zavala J.S.:
Departamento de Bioquímica, Instituto Nacional de Cardiología, México D.F., Mexico
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