Hydrolysis of 4-methylumbelliferyl N-acetyl-chitooligosaccharides catalyzed by human lysozyme.
Binding of N-acetyl-chitotetraose, 4-methylumbelliferyl chitotrioside ((GlcNAc)3-MeU), and 4-methylumbelliferyl chitotetraoside ((GlcNAc)4-MeU) to human lysozyme [EC 3.2.1.17] was studied by fluorescence measurement. Hydrolysis of (GlcNAc)3-MeU and (GlcNAc)4-MeU catalyzed by human lysozyme was studied by measuring the release of 4-methylumbelliferone fluorimetrically and the kinetic constants were determined in the pH range of 2 to 8 at 0.1 ionic strength and 42 degrees C. On the basis of binding and kinetic data, it was shown that (GlcNAc)3-MeU binds mainly to subsites A to D with the terminal MeU group bound to subsite D (nonproductive binding) and that (GlcNAc)4-MeU binds to subsites A to E (productive binding) and subsites A to D with the nonreducing sugar residue extending beyond subsite A (nonproductive binding). The pH dependences of the kinetic constants for hydrolysis of (GlcNAc)3-MeU and (GlcNAc)4-MeU were analyzed assuming that nonproductive binding occurs competitively, that an ionizable group in addition to the catalytic groups (Asp 52 and Glu 35) participates in the catalysis, and that the molecular species with ionized Asp 52 and protonated Glu 35 is active. Analyses of the kinetic constants for (GlcNAc)3-MeU and (GlcNAc)4-MeU both gave the same pK values of the catalytic groups (pK52 = 3.6(3) and pK35 = 6.6(8) at 0.1 ionic strength and 42 degrees C). These pK values were very close to the values determined previously by spectroscopic methods in our laboratory (Kuramitsu et al. (1974) J. Biochem. 76, 671-683; (1978) ibid. 83, 159-170; (1980) ibid. 87, 771-778).
D002686:Chitin / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism*; D007700:Kinetics; D009113:Muramidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014468:Umbelliferones / Q000378:metabolism*
C015366:4-methylumbelliferyl-N-acetyl-chitotrioside; C027049:4-methylumbelliferyl-N-acetylchitotetraoside; D009844:Oligosaccharides; D014468:Umbelliferones; D002686:Chitin; D006923:Hymecromone; D009113:Muramidase