Thermodynamic integration calculations of binding free energy difference for Gly-169 mutation in subtilisin BPN′

The binding free energy difference for the Gly-169 → Ala-169 (G169A) mutation in subtilisin BPN′ complexed with a tripeptide substrate analogue is explored using the thermodynamic integration approach. The structure of the mutant enzyme–substrate complex obtained from free energy simulation is in good agreement with experimental X-ray refinement. The near perfect reversibility is obtained in the present work for ensuring the correctness of the free energy calculations. The results of the binding free energy difference are close to similar experimental data. © 1993 Wiley-Liss, Inc.