Enzymatic Flexibility and Reaction Rate: A QM/MM Study of HIV‑1 Protease

The relevance of conformational fluctuations on enzyme rates has been a matter of debate for decades. Single molecule experiments have detected variations on the catalytic rates between different enzyme molecules, and within the same enzyme molecule, in a time scale larger than turnover. Computational methods can detect different energy barriers, induced by thermal conformational fluctuations, at a microscopic time scale, several orders of magnitude faster than the turnover rate of the fastest enzyme. Others have observed these barrier fluctuations, but few computational studies have dissected them in detail and tried to understand their origins and consequences. For this purpose, we studied the first step of the reaction catalyzed by HIV-1 Protease, starting from 40 different conformations. We found activation free energies ranging from 14.5 to 51.3 kcal·mol–1. The calculated apparent barrier is 16.5 kcal·mol–1, which is very close to the experimental value of 15.9 kcal·mol–1 for product release. These fluctuations are determinant to the overall rate, and these are correlated to specific structural changes. The effect of each enzymatic conformation on the stabilization of the transition state can be explained by the electrostatic interaction of every protein residue with the flow of net electronic density (negative charge) from the reactants to the transition state.