Site specific point mutation changes specificity: a molecular modeling study by free energy simulations and enzyme...

We have theoretically and experimentally studied the binding of two different ligands to wild-type ribonuclease T1 (RNT1) and to a mutant of RNT1 with Glu-46 replaced by Gln. The binding of the natural substrate 3'-GMP has been compared with the binding of a fluorescent probe, 2-aminopurine 3'-monophosphate (2AP), and relative free energies of binding of these ligands to the mutant and the wild-type (wt) enzyme have been calculated by free energy perturbation methods. The free energy perturbations predict that the mutant RNT1-Gln-46 binds 2AP better than 3'GMP, in agreement with experiments on dinucleotides. Four free energy perturbations, forming a closed loop, have been performed to allow the detection of systematic errors in the simulation procedure. Because of the larger number of atoms involved, it was necessary to use a much longer simulation time for the change in the protein, i,e., the perturbation from Glu to Gln, than in the perturbation from 3'-GMP to 2AP. Finally the structure of the binding site is analyzed for understanding differences in catalytic speed and binding strength.