Thermodynamics of inhibitor binding to the catalytic site of glucoamylase from Aspergillus niger determined by displacement titration calorimetry

Bent W. Sigurskjold, Carolyn R. Berland, Birte Svensson

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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 pK(a) 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). All ligands bind with remarkably favorable contributions from entropy, indicating good structural complementarity with efficient solvent displacement in complex formation.
Original languageEnglish
JournalBiochemistry
Volume33
Issue number33
Pages (from-to)10191-10199
ISSN0006-2960
DOIs
Publication statusPublished - 1994
Externally publishedYes

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