title: Role of acidic amino acid residues in chitooligosaccharide-binding to Streptomyces sp. N174 chitosanase.
abstract: We examined the oligosaccharide binding to Streptomyces sp. N174 chitosanase by fluorescence spectroscopy. By means of the tryptophan fluorescence quenching, the oligosaccharide binding abilities were evaluated using the three mutant enzymes (D57A, E197A, and D201A). The enzymatic activities of the mutant enzymes were 0.5%, 20.0%, and 38.5% of that of the wild type, respectively. Scatchard plot obtained for the wild type enzyme showed a biphasic profile, suggesting that the oligosaccharide binds to the chitosanase with two different binding sites (the high affinity site and the low affinity site). In contrast, Scatchard plot for E197A exhibited a monophasic profile, in which the slope of the line corresponds to that for the low affinity binding of the wild type enzyme. A monophasic profile was also obtained for D201A, but the slope of the line was similar to that of the high affinity binding. Thus, we conclude that Glu197 and Asp201 are responsible for oligosaccharide binding at the high affinity site and the low affinity site, respectively, which correspond to the (-n) subsites and the (+n) subsites (n=1, 2, and 3). The fluorescence quenching was very weak in D57A, suggesting a strong contribution of this residue to the oligosaccharide binding.
authors.affiliation: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Katsumi
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Lacombe-Harvey
authors.forename: Hugo
authors.initials: H
authors.lastname: Tremblay
authors.forename: Ryszard
authors.initials: R
authors.lastname: Brzezinski
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic / Q000502:physiology*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D024342:Amino Acids, Acidic; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; C019868:chitosanase