Steady-state kinetic and calorimetric studies on the binding of Aspergillus niger glucoamylase with gluconolactone, 1-deoxynojirimycin, and beta-cyclodextrin.
Binding equilibria of Aspergillus niger glucoamylase with its several ligands were observed to analyze the binding modes of the ligands. Steady-state kinetic studies using p-nitrophenyl alpha-glucoside as a substrate showed that 1-deoxynojirimycin, which is a mixed type inhibitor for Rhizopus glucoamylase, was a competitive type inhibitor bound at the active site of the enzyme, but gluconolactone, which is also a mixed type inhibitor for Rhizopus glucoamylase, was a non-competitive type inhibitor forming a nonproductive ternary complex with the enzyme and the substrate. beta-Cyclodextrin, which binds to the starch-binding domain of the enzyme, did not inhibit the enzyme activity, showing that there was no interaction between the catalytic domain and the starch-binding domain for the binding of the substrate and beta-cyclodextrin. Isothermal titration calorimetry showed that one 1-deoxynojirimycin molecule and two beta-cyclodextrin molecules bind to the catalytic domain and the starch-binding domain of the enzyme, respectively, and there is no significant interaction between the binding of these ligands.
Tanaka
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Department of Chemistry, Faculty of Education, Mie University, Japan.
D017485:1-Deoxynojirimycin / Q000494:pharmacology*; D001234:Aspergillus niger / Q000201:enzymology*; D002151:Calorimetry; D003505:Cyclodextrins / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005087:Glucan 1,4-alpha-Glucosidase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D005942:Gluconates / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry; D007700:Kinetics; D007783:Lactones; D011485:Protein Binding; D013816:Thermodynamics; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D004791:Enzyme Inhibitors; D005942:Gluconates; D007527:Isoenzymes; D007783:Lactones; D047392:beta-Cyclodextrins; D017485:1-Deoxynojirimycin; D005087:Glucan 1,4-alpha-Glucosidase; C031215:betadex; C010730:beta-glucono-1,5-lactone