A β-galactosidase from pea seeds (PsBGAL): purification, stabilization, catalytic energetics, conformational heterogeneity, and its significance

Abstract

A basic glycosylated β-galactosidase (PsBGAL) has been purified from pea seeds by 910-fold with a specific activity of 77.33 μmol min-1 mg-1 protein. The purified enzyme is an electrophoretically homogeneous protein consisting of a single protein band with an apparent Mr of 55 kDa, while the deglycosylated enzyme has a Mr of 54.2 kDa on SDS-PAGE under reducing conditions. According to MALDI-TOF measurements of the 55 kDa band, the enzyme showed a homology with BGAL from other sources present in the SWISS-PROT database, while it showed no resemblance to any lectin. The N-terminal sequence of PsBGAL was determined as TIECK and showed a resemblance to BGAL from Arabidopsis thaliana (Q93Z24). The enzyme showed an unique property of multiple banding patterns on SDS-PAGE at 20 mA current, with tryptic digests of all bands having similar m/z values (using MALDI-TOF) while it showed only a single band at 10 mA current. PsBGAL is effectively compartmentalized during seed maturation inside vacuoles (pH ∼ 5). The enzyme is capable of hydrolyzing pea seed xyloglucan, and it may be involved in modifying the cell wall architecture during seedling growth and development. The enzyme has a protonated carboxyl group at its active site as observed by ionization constant, thermodynamics, and chemical modification studies. © 2009 American Chemical Society.