IIBYT   23944
INSTITUTO DE INVESTIGACIONES BIOLOGICAS Y TECNOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The use of isothermal titration calorimetry to measure enzyme kinetics in molecular crowding systems.
Autor/es:
NOLAN MV; SANCHEZ JM; PERILLO MA
Lugar:
San Javier, Tucumán
Reunión:
Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
The cellular cytoplasm is composed
of different molecular species that generate a crowded environment.
Macromolecules occupy an important volume fraction that cannot be occupied by
other molecules. As a consequence of this, macromolecules undergo steric and
diffusional restrictions that affect their properties. The effects of crowding
on biochemical reaction rates are complex because although crowding reduces
diffusion, it increases thermodynamic activities.
b-galactosidase (b-Gal) is an enzyme that catalizes lactose hydrolysis. It has been
extensively studied because of its nutritional, biotechnological and
therapeutic impact. Not only in the cellular milieu but also in situations of
technological interest the activity of b-Gal has to be evaluated in crowded systems.
Determination of enzyme kinetics is
traditionally measured with a variety of methods including spectroscopy. Using
spectral methods when measuring enzyme kinetics in crowded solutions are
limited due to scattering and/or absorption of the light. The use of
calorimetry to measure the rate of enzymatic reactions appears as a good option
in these cases. Isothermal titration calorimetry (ITC) measures the heat-flow
associated with a given chemical reaction. The rate of the enzymatic reaction
is proportional to this heat flow, and thus measured as the primary
experimental observable. Since practically all reactions produce or consume
heat, enzyme activity can be monitored in real time without the need of probes,
substrate analogues, etc.
The aims of this study were: i) to
investigate the effect of macromolecular crowding on the kinetics of hydrolysis
of o-nitrophenyl galactopyranoside (ONPG) catalyzed by b-Gal, ii) to
compare ONPG hydrolysis data obtained by UV-visible spectroscopy and ITC, to
validate the ITC technique to study enzyme kinetics. Molecular crowding was
simulated with solutions of polyethylene glycol at different concentrations.
The results showed that Vmax was not affected, while the affinity of
the enzyme (KM) suffered a significant decrease when molecular
crowding was increased. It could be due to the restriction imposed by
diffusional high molecular density present in these conditions. The results
obtained by the two techniques (spectroscopy and ITC) were not significantly
different, allowing us to validate the use of ITC as a technique to study the
kinetics of reactions mediated by enzymes both in diluted and in crowded
solutions
Acknowledgements: CONICET,
SeCyT-UNC, Mincyt-Prov.Córdoba y Foncyt.
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