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, T he mutants studied comprise a group of single amino acid replacements in conserved regions near the active site of the enzyme. For each muta nt we have determined both the affinities for the two inhibitors and t he thermodynamic state functions for binding using titration microcalo rimetry, We find that acarbose binds to all the mutants with a wide ra nge of binding constants (10(4) < K-a < 10(13) M(-1)). In contrast, 1- deoxynojirimycin shows either binding at near wild-type affinity (K-a congruent to 10(4) M(-1)) or no detectable binding. The changes in the affinities of the mutant enzymes are rationalized in terms of the kno wn three-dimensional structure of the wild-type enzyme with subsites 1 , 2, and 3 being important for acarbose binding while only subsite 1 i s critical for 1-deoxynojirimycin binding. In most of the mutants stud ied the magnitudes of the enthalpies and the entropies of binding of t he mutant: enzymes differed from those of the wild-type enzyme with th e mutant enzymes having a relatively large portion of their binding en ergy composed of enthalpy and a relatively small proportion composed o f entropy. The pattern of changes in the enthalpy and entropy is hypot hesized to be due to changes in the structural complementarity of the binding pocket and the inhibitor.