TAILIEUCHUNG - Báo cáo khoa học: Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase Mechanism and kinetics of inhibition by acarbose and cyclodextrins

Cyclodextrin glycosyltransferase (CGTase) uses ana-retain-ingdoubledisplacementmechanismtocatalyze threedistinct transglycosylation reactions. To investigate these reactions as catalyzed by the CGTase fromThermoanaerobacterium thermosulfurigenesthe enzyme was overproduced (8 mgÆL )1 culture) usingBacillus subtilisas a host. Detailed analysis revealed that the three reactions proceed via different kinetic mechanisms. | Eur. J. Biochem. 270 155-162 2003 FEBS 2003 doi Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase Mechanism and kinetics of inhibition by acarbose and cyclodextrins Hans Leemhuis1 Bauke W. Dijkstra2 and Lubbert Dijkhuizen1 1 Department of Microbiology Groningen Biomolecular Sciences and Biotechnology Institute GBB University of Groningen NN Haren the Netherlands 2BIOSON Research Institute and Laboratory of Biophysical Chemistry GBB University of Groningen AG Groningen the Netherlands Cyclodextrin glycosyltransferase CGTase uses an a-retain-ing double displacement mechanism to catalyze three distinct transglycosylation reactions. To investigate these reactions as catalyzed by the CGTase from Thermoanaerobacterium thermosulfurigenes the enzyme was overproduced 8 mg L-1 culture using Bacillus subtilis as a host. Detailed analysis revealed that the three reactions proceed via different kinetic mechanisms. The cyclization reaction cyclodextrin formation from starch is a one-substrate reaction whereas the other two transglycosylation reactions are two-substrate reactions which obey substituted enzyme mechanism kinetics disproportionation reaction or ternary complex mechanism kinetics coupling reaction . Analysis of the effects of acarbose and cyclodextrins on the disproportionation reaction revealed that cyclodextrins are competitive inhibitors whereas acarbose is a mixed type of inhibitor. Our results show that one molecule of acrirbose binds either in the active site of the free enzyme or at a secondary site of the enzyme-substrate complex. The mixed inhibition thus indicates the existence of a secondary sugar binding site near the active site of T. thermosulfurigenes CGTase. Keywords CGTase transglycosylation enzyme mechanism acarbose inhibition. The a-amylase family or glycoside hydrolase family 13 1 2 is a large family of starch processing enzymes which form a wide variety of oligosaccharide products 3 4 via .

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