Trapping of a covalent enzyme intermediate in the reaction of Bacillus macerans cyclomaltodextrin glucanyltransferase with cyclomaltohexaose

Soo Bok Lee, John F. Robyt

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)47-53
Number of pages7
JournalCarbohydrate Research
Volume336
Issue number1
DOIs
Publication statusPublished - 2001

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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