Sucrose- and Dextran-binding Sites of Dextransucrase Analyzed by Chemical Modification with o-Phthalaldehyde

概要

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The treatment of Leuconostoc mesenteroides B-512F dextransucrase with 1.25 mNi o-phthalaldehyde (OPA) inactivated the enzyme almost completely within 30 min. The addition of substrates dextran T-10 or sucrose retarded the enzyme inactivation by OPA. The addition of sucrose-monocaprate, an analog of sucrose, also retarded the inactivation. Differential modification of dextransucrase by OPA was conducted in the presence of sucrose, dextran T-10, or sucrosemonocaprate. Modified enzymes were digested by trypsin, and the resultant peptides were separated by HPLC. All of the peptides containing lysine residues protected by the ligands had homologies to the catalytic domain of streptococcal glucosyltransferases (GTFs), and these regions in GTFs were apart from the catalytic aspartic acid. Two peptides which contained sucrose-protected lysine were located in the direction of the amino terminal from the catalytic aspartate. Those peptides were also protected from the OPA modification by dextran and there were two more dextran-protected peptides which were close to the glucan-binding domain. Hydroxyl amino acids were frequently found in those substrate-protected peptides, indicating that the peptides originated from the sugar-affinity sites.
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