| Abstract: | An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH4)2SO4 precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, Km 0.29 mM, Vmax 2.56 μkat mg−1) and pNP-β-d-GalNAc (optimum pH 4.4, Km 0.27 mM, Vmax 2.50 μkat mg−1). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The Kis for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)2 and pNP-β-d-(GlcNAc)3 than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)2 (optimum pH 5.0) and (GlcNAc)3−6 (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)n were predominantly (GlcNAc)n−1 and GlcNAc. |
