IIBYT   23944
INSTITUTO DE INVESTIGACIONES BIOLOGICAS Y TECNOLOGICAS
Unidad Ejecutora - UE
artículos
Título:
PEG-induced molecular crowding leads to a relaxed conformation, higher thermal stability and lower catalytic efficiency of Escherichia coli beta-galactosidase.
Autor/es:
VERONICA NOLAN; JULIETA M. SÁNCHEZ,; MARÍA A. PERILLO
Revista:
COLLOIDS AND SURFACES B-BIOINTERFACES
Editorial:
ELSEVIER SCIENCE BV
Referencias:
Lugar: Amsterdam; Año: 2015 vol. 136 p. 1202 - 1206
ISSN:
0927-7765
Resumen:
Enzymatic activities were historically assayed in dilutesolutions where molecular crowding, molecular confinement and theirconsequences were not taken into account. Here we report howmacromolecular crowding tunes catalytic parameters for the tetrameric β-Galactosidase from Escherichia coli, β-Gal. We detected increases in KM(weaker substrate binding) and a nonlinear variation in Vmax, with aminimum at 25 %W/P of the crowding agent (polyethyleneglycol molecularmass 6000, PEG6000) resulting in a linear decrease in the catalyticefficiency (kcat/KM) within the whole [PEG6000] range tested). Presenceof crowding agent affected β-Gal structural content and increased itsthermal resistance. Steady state fluorescence and Fourier transformedinfrared spectroscopic observations are compatible with crowding-induceddisordering and restricted internal dynamics as a result of excludedvolume and solvent structuring effects. This leads to a non-optimalsubstrate-binding site and a less conformationally strained protein.