The Role Of Solvent Effect On The Ligand Binding Properties Of The Thermostable Variants Of Aminoglycoside Nucleotidyltransferase 4(-)(Ant4) Seda Kocaman

The aminoglycoside nucleotidyltransferase 4’ (ANT) is a homodimeric enzyme that detoxifies a large number of aminoglycoside antibiotics by nucleotidylating at the C4’-OH site. Two thermostable variants for this enzyme show only a single amino acid changes in their primary amino acid sequences (T130K, D80Y). It is not known how single residue replacements, which are distant from active site and monomer-monomer interface, result in various degrees of changes on thermostability of the enzyme. Thermodynamic parameters of the binary enzyme-aminoglycoside complexes, however, show highly significant differences. The data, acquired in H2O and D2O in this work by isothermal titration calorimetry also demonstrate that solvent reorganization upon ligand binding show large differences between the two variants. The heat capacity change (ΔCp) also show antibiotic-dependent differences between the two variants. Thus, data shown in this work suggest that thermodynamics of ligand-protein interactions and solvent effects may be among the molecular parameters that separate thermophilic proteins from simply those that are thermostable but otherwise identical to the mesophilic counterparts.