title: Trapping of a covalent enzyme intermediate in the reaction of Bacillus macerans cyclomaltodextrin glucanyltransferase with cyclomaltohexaose.
abstract: The mechanism of catalysis of Bacillus macerans cyclomaltodextrin glucanyltransferase (CGTase, EC 2.4.1.19) was studied by trapping and isolating a covalent-enzyme intermediate. CGTase catalyzes an acceptor or coupling reaction between cyclomaltohexaose and a carbohydrate acceptor such as D-glucose. CGTase was incubated with 3H-labeled cyclomaltohexaose in the absence of any added acceptor. After 30 s of reaction, the enzyme was rapidly denatured and precipitated by the addition of 10% trifluoroacetic acid (TFA). Extensive washing of the precipitated protein showed retention of radioactivity with the protein. The precipitate was dissolved in 0.1 M TFA, containing 6 M urea and passed over a BioGel P-10 column. The protein fraction retained 95% of its original radioactivity. The reaction with [3H]cyclomaltohexaose was also stopped by the addition of TFA to give an inactive enzyme at pH 2.5. The enzyme was separated from unreacted cyclomaltohexaose on a BioGel P-10 column and was shown to be radioactive. When the radioactive protein fraction was rechromatographed on BioGel P-10, it retained 100% of the label. These results demonstrate the formation of a covalent carbohydrate-enzyme intermediate in the reactions catalyzed by CGTase.
authors.affiliation: Laboratory of Carbohydrate Chemistry and Enzymology, Iowa State University, 4254 Molecular Biology Building, Ames, IA 50011, USA.
authors.forename: S B
authors.initials: SB
authors.lastname: Lee
authors.forename: J F
authors.initials: JF
authors.lastname: Robyt
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D002384:Catalysis; D011232:Chemical Precipitation; D002845:Chromatography; D003505:Cyclodextrins / Q000737:chemistry / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D011849:Radioactive Tracers; D014316:Tritium; D047391:alpha-Cyclodextrins
chemical_list: D003505:Cyclodextrins; D011849:Radioactive Tracers; D047391:alpha-Cyclodextrins; D014316:Tritium; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; C032613:alpha-cyclodextrin