title: Thermodynamics of inhibitor binding to the catalytic site of glucoamylase from Aspergillus niger determined by displacement titration calorimetry.
abstract: The binding of different inhibitors to glucoamylase G2 from Aspergillus niger and its temperature and pH dependencies have been studied by titration calorimetry. The enzyme binds the inhibitors 1-deoxynojirimycin and the pseudo-tetrasaccharide acarbose with association constants of 3 x 10(4) and 9 x 10(11) M-1, respectively, at 27 degrees C. The binding free energy for both ligands is remarkably temperature-invariant in the interval from 9 to 54 degrees C as the result of large compensating changes in enthalpy and entropy. Acarbose and 1-deoxynojirimycin bound with slightly different free energy-pH profiles, with optima at 5.5 and 5.5-7.0, respectively. Variations in delta H degrees and T delta S degrees as a function of pH were substantially larger than variations in delta G degrees in a partly compensatory manner. Two titratable groups at or near subsite 1 of the catalytic site were found to change their pKa slightly upon binding. The hydrogenated forms of acarbose, D-gluco- and L-ido-dihydroacarbose, bind with greatly reduced association constants of 3 x 10(7) and 2 x 10(5) M-1, respectively, and the pseudo-disaccharide methyl acarviosinide, lacking the two glucose units at the reducing end compared to acarbose, has a binding constant of 8 x 10(6) M-1; these values all result from losses in both enthalpy and entropy compared to acarbose. Three thio analogues of the substrate maltose, methyl alpha- and beta-4-thiomaltoside and methyl alpha-4,5'-dithiomaltoside, bind with affinities from 3 x 10(3) to 6 x 10(4) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
authors.forename: B W
authors.initials: BW
authors.lastname: Sigurskjold
authors.forename: C R
authors.initials: CR
authors.lastname: Berland
authors.forename: B
authors.initials: B
authors.lastname: Svensson
mesh_terms: D017485:1-Deoxynojirimycin / Q000378:metabolism; D020909:Acarbose; D001234:Aspergillus niger / Q000201:enzymology; D001665:Binding Sites; D002151:Calorimetry; D005087:Glucan 1,4-alpha-Glucosidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008320:Maltose / Q000031:analogs & derivatives / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism
chemical_list: D014312:Trisaccharides; D017485:1-Deoxynojirimycin; D008320:Maltose; D005087:Glucan 1,4-alpha-Glucosidase; D020909:Acarbose