Purification and enzymatic characterization of tobacco leaf β-N-acetylhexosaminidase

• Published in: Biochimie 2014-12, Vol.107, p.263-269
• Main Authors: Ryšlavá, Helena, Valenta, Robert, Hýsková, Veronika, Křížek, Tomáš, Liberda, Jiří, Coufal, Pavel
• Format: Article
• Language: English
• Subjects: Acetylgalactosamine - analogs & derivatives; Acetylgalactosamine - metabolism; Acetylglucosamine - analogs & derivatives; Acetylglucosamine - metabolism; Acetylglucosamine - pharmacology; beta-N-Acetylhexosaminidases - antagonists & inhibitors; beta-N-Acetylhexosaminidases - chemistry; beta-N-Acetylhexosaminidases - isolation & purification; beta-N-Acetylhexosaminidases - metabolism; Capillary electrophoresis; Chitooligomers; Disaccharides - metabolism; Enzyme Inhibitors - pharmacology; Hydrogen-Ion Concentration; Hydrolysis; Inhibition by excess substrate; Kinetics; Molecular Weight; Nicotiana - enzymology; Plant Leaves - enzymology; Substrate Specificity; β-N-acetylhexosaminidase
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2014.09.006

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. •β-N-acetylhexosaminidase was purified from tobacco leaves; the native molecule is a glycoprotein of Mr 280 000.•This enzyme was able to hydrolyze pNP-GlcNAc, pNP-GalNAc, (GlcNAc)2, (GlcNAc)3, and pNP-(GlcNAc)2.•pNP-GlcNAc caused the inhibition by excess of this substrate, other studied substrates showed classical hyperbolic kinetics.•GlcN, GlcNAc, GalN, and GalNAc were inhibitors; the inhibition constants and type of inhibition were determined.•Possible role of studied enzyme could be in the response against pathogens and glycoprotein processing.