Trapping of a covalent enzyme intermediate in the reaction of Bacillus macerans cyclomaltodextrin glucanyltransferase with cyclomaltohexaose.
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.
Lee
S B
SB
Laboratory of Carbohydrate Chemistry and Enzymology, Iowa State University, 4254 Molecular Biology Building, Ames, IA 50011, USA.
Robyt
J F
JF
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
D003505:Cyclodextrins; D011849:Radioactive Tracers; D047391:alpha-Cyclodextrins; D014316:Tritium; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; C032613:alpha-cyclodextrin