Evidence for the presence of essential histidines on cyclodextrin glucanotransferase from Bacillus macerans

Cyclodextrin glucanotransferase (CGTase) from Bacillus macerans was rapidly inactivated by diethyl pyrocarbonate (DEP). Kinetic studies demonstrated that a reversible DEP-enzyme complex was not formed before the inactivation process. The second-order rate constant for inactivation of CGTase, calcula...

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Bibliographic Details
Published inBiotechnology and applied biochemistry Vol. 19; pp. 85 - 92
Main Authors Jeang, C.L, Lin, Y.W
Format Journal Article
LanguageEnglish
Published 01.01.1994
Subjects
binding sites
cyclomaltodextrins
enzyme activity
histidine
kinetics
maltodextrins
Paenibacillus macerans
transferases
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Summary:Cyclodextrin glucanotransferase (CGTase) from Bacillus macerans was rapidly inactivated by diethyl pyrocarbonate (DEP). Kinetic studies demonstrated that a reversible DEP-enzyme complex was not formed before the inactivation process. The second-order rate constant for inactivation of CGTase, calculated to be 1073 M-1.min-1, was pH-dependent, with a pK of approx 6.3. The loss of enzymic activity was correlated with the acylation of histidine residues. Activity could be restored by addition of neutral hydroxylamine, and protection was afforded by the substrates alpha-, beta- and gamma-cyclodextrins. The u.v.-absorption spectra of the DEP-modified enzymes revealed an absorption maximum at 237-242 nm, characteristic for N-ethoxycarbonylhistidine. Inactivation of CGTase with DEP required modification of six histidine residues/molecule of the enzyme. Spectral analysis of CGTase obtained under different modification conditions indicated that alpha-cyclodextrin prevented the modification of four crucial histidine residues. The overall results provide evidence that four active-site histidine residues are involved in the catalytic reaction mechanism of CGTase.
ISSN:0885-4513
1470-8744