title: Purification and enzymatic characterization of tobacco leaf β-N-acetylhexosaminidase.
abstract: The kinetic properties of β-N-acetylhexosaminidase purified from tobacco (Nicotiana tabacum L.) leaves have been investigated. In addition to chromogenic pNP derivates, N,N'-diacetylchitobiose and N,N',N″-triacetylchitotriose were also used as substrates of β-N-acetylhexosaminidase. The highest reaction rate and the affinity for the substrate were observed for pNP-GlcNAc; however, an excess of this substrate inhibits the reaction. The reaction rate with pNP-GalNAc as the substrate was found to be about 85% of that obtained with pNP-GlcNAc. The hydrolysis of acetylated chitooligomers by β-N-acetylhexosaminidase followed by separation and quantification using capillary electrophoresis was slower compared to pNP-GlcNAc. The pH optimum of β-N-acetylhexosaminidase for individual substrates was found at 4.3-5.0 and the temperature optimum was 50-55 °C. Gel permeation chromatography and red native electrophoresis determined the relative molecular weight as 280 000 and the isoelectric point as 5.3. The inhibition of β-N-acetylhexosaminidase by monosaccharides GlcN, GalN, GlcNAc, GalNAc in combination with substrates pNP-GlcNAc and pNP-GalNAc was studied and the type of inhibition and the inhibition constants were determined.
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2 128 40, Czech Republic. Electronic address: helena.ryslava@natur.cuni.cz.
authors.forename: Helena
authors.initials: H
authors.lastname: Ryšlavá
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2 128 40, Czech Republic.
authors.forename: Robert
authors.initials: R
authors.lastname: Valenta
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2 128 40, Czech Republic.
authors.forename: Veronika
authors.initials: V
authors.lastname: Hýsková
authors.affiliation: Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2 128 40, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Křížek
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2 128 40, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Liberda
authors.affiliation: Department of Analytical Chemistry, Faculty of Science, Charles University, Hlavova 2030, Prague 2 128 40, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Coufal
mesh_terms: D000116:Acetylgalactosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D004187:Disaccharides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D018515:Plant Leaves / Q000201:enzymology; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology*; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004187:Disaccharides; D004791:Enzyme Inhibitors; C014123:4-nitrophenyl-2-acetamido-2-deoxygalactopyranoside; C010913:N,N-diacetylchitobiose; C014124:4-nitrophenyl-N-acetyl-2-deoxyglucopyranoside; D001619:beta-N-Acetylhexosaminidases; D000116:Acetylgalactosamine; D000117:Acetylglucosamine
keywords: Capillary electrophoresis; Chitooligomers; Inhibition by excess substrate; β-N-acetylhexosaminidase