Purification and substrate specificity of an endo-beta-N-acetylglucosaminidase from pea (Pisum sativum) seeds.
An endo-beta-N-acetylglucosaminidase was purified to homogeneity from the seeds of pea (Pisum sativum). The molecular mass of the purified enzyme was estimated to be 41,669 Da by MALDI-TOF MS analysis and its isoelectric point to be 4.3 by isoelectric focusing. The enzyme was stable at pH 4-7 and at 25-50 degrees C, and had the highest activity toward Man6GlcNAc2-PA at pH around 7.0. Oligomannose type sugar chains (Man9-6GlcNAc2-PA) and a hybrid type sugar chain (GlcNAc1Man5GlcNAc2-PA) were most favored substrates followed by Man5GlcNAc2-PA, Man3GlcNAc2-PA, and GlcNAc2Man3GlcNAc2-PA, but xylose-containing sugar chains (Man4-3Xyl1GlcNAc2-PA and Man3Fuc1Xyl1GlcNAc2-PA) or a biantennary complex type sugar chain (Gal2GlcNAc2Man3GlcNAc2-PA) could not be hydrolyzed by the enzyme. The Km values of the enzyme for Man5GlcNAc2-PA, Man6GlcNAc2-PA, and Man9GlcNAc2-PA were 0.40 mM, 0.25 mM, and 0.32 mM, respectively.
Kimura
Y
Y
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Japan.
Iwata
K
K
Sumi
Y
Y
Takagi
S
S
D001483:Base Sequence; D002412:Cations; D003902:Detergents / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018532:Pisum sativum / Q000201:enzymology*; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature
D002412:Cations; D003902:Detergents; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase