Thermodynamics of inhibitor binding to mutant forms of glucoamylase from Aspergillus niger determined by isothermal titration calorimetry.
We have investigated the binding of mutant forms of glucoamylase from Aspergillus niger to the inhibitors 1-deoxynojirimycin and acarbose. The mutants studied comprise a group of single amino acid replacements in conserved regions near the active site of the enzyme. For each mutant we have determined both the affinities for the two inhibitors and the thermodynamic state functions for binding using titration microcalorimetry. We find that acarbose binds to all the mutants with a wide range of binding constants (10(4) < Ka < 10(13) M-1). In contrast, 1-deoxynojirimycin shows either binding at near wild-type affinity (Ka approximately equal to 10(4) M-1) or no detectable binding. The changes in the affinities of the mutant enzymes are rationalized in terms of the known three-dimensional structure of the wild-type enzyme with subsites 1, 2, and 3 being important for acarbose binding while only subsite 1 is critical for 1-deoxynojirimycin binding. In most of the mutants studied the magnitudes of the enthalpies and the entropies of binding of the mutant enzymes differed from those of the wild-type enzyme with the mutant enzymes having a relatively large portion of their binding energy composed of enthalpy and a relatively small proportion composed of entropy. The pattern of changes in the enthalpy and entropy is hypothesized to be due to changes in the structural complementarity of the binding pocket and the inhibitor.
Berland
C R
CR
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Sigurskjold
B W
BW
Stoffer
B
B
Frandsen
T P
TP
Svensson
B
B
D017485:1-Deoxynojirimycin / Q000378:metabolism; D020909:Acarbose; D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D002240:Carbohydrate Sequence; D005087:Glucan 1,4-alpha-Glucosidase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013816:Thermodynamics; D014312:Trisaccharides / Q000378:metabolism
D014312:Trisaccharides; D017485:1-Deoxynojirimycin; D005087:Glucan 1,4-alpha-Glucosidase; D020909:Acarbose