A beta-galactosidase from pea seeds (PsBGAL): purification, stabilization, catalytic energetics, conformational heterogeneity, and its significance.
A basic glycosylated beta-galactosidase (PsBGAL) has been purified from pea seeds by 910-fold with a specific activity of 77.33 mumoL min(-1) mg(-1) protein. The purified enzyme is an electrophoretically homogeneous protein consisting of a single protein band with an apparent M(r) of 55 kDa, while the deglycosylated enzyme has a M(r) 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 approximately 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.
Dwevedi
Alka
A
School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, UP-221005, India.
Kayastha
Arvind M
AM
D002384:Catalysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018532:Pisum sativum / Q000737:chemistry / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D012639:Seeds / Q000737:chemistry / Q000201:enzymology; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D010940:Plant Proteins; D001616:beta-Galactosidase