Bovine N-acetyl-beta-D-glucosaminidase: affinity purification and characterization of its active site with nitrogen containing analogs of N-acetylglucosamine.
Two N-acetylglucosaminidases were isolated from bovine kidney with a three step procedure featuring affinity purification on 2-acetamido-1,2,5-trideoxy-1,5-iminoglucitol (2-acetamido-1,2-dideoxynojirimycin, II). The major isoenzyme, Hex A, is an alpha, beta hetero-dimer (57 and 52 kDa) with isoelectric points from pH 5.3 to 6.6 and comprised about 80% of the total activity. Its kinetic properties with respect to discrimination between N-acetylglucosaminide, N-acetylgalactosaminide and the corresponding 6-sulfate ester were similar to human hexosaminidase A. The minor isoenzyme, Hex B, a homodimer, isoelectric points 7.0 to 7.4, was similar to Hex A but was without detectable activity with methylumbelliferyl-N-acetyl-beta-glucosaminide-6-sulfate. Inhibition studies with Hex A were carried out with 2-acetamido-2,5-dideoxy-1,5-imino-D-glucopyranose (2-acetamido-2-deoxynojirimycin, (1), the corresponding 1,5-lactam (III), with II and its N,N-dimethyl derivative, and with 2-acetamido-2-deoxy-D-glucono-1,5-lactone (IV). In comparison with N-acetylglucosamine (Ki 1.9 mM) Hex A was inhibited 10(6)-fold better by I, 2600-fold better by II, 2900-fold better by III, and 55,000-fold better by IV. A slow approach to the inhibition equilibrium was observed with I and IV. For IV and Hex A it is the first example of a slow inhibition of a glycoside hydrolase by the corresponding glycono-1,5-lactone. The pH-dependence of Ki for the permanently cationic N,N-dimethyl II (15.4 microM (pH 3.5) to 0.47 microM (pH 7.0)) indicated that formation of the enzyme inhibitor complex is governed by deprotonation of a group with pKa 5.0. The results are discussed with respect to structural features and water accessibility of the active site.
Legler
G
G
Institut für Biochemic, Universität zu Köln, F.R.G.
Lüllau
E
E
Kappes
E
E
Kastenholz
F
F
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D000118:Acetylglucosaminidase / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D013379:Substrate Specificity
D007527:Isoenzymes; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine