The peptide bond rupture mechanism in the serine proteases: an in silico study based on sequential scale models

Given the importance of serine proteases for biochemical processes, we have studied the peptide bond rupture mechanism using three sequential scale models as representations of the KLK5 enzyme (a protein overexpressed in ovarian cancer). The first model contains the basic functional groups of the residues that conform to the catalytic triad present in serine proteases; the second model contains some additional residues and, finally, the last representation includes all atoms of the KLK5 protein together with 10.000 explicit water molecules. This separation into three scale models allows us to separate the intrinsic reactivity of the catalytic triad from the process taking place in the enzyme. The methodologies employed in this work include full DFT calculations with a dielectric continuum in the first two models and a multi-level setup with a Quantum Mechanics/Molecular Mechanics (QM/MM) partition in the whole protein system. Our results show that the peptide-bond rupture mechanism is a stepwise process involving two proton transfer reactions. The rate-determining step is the second proton transfer from the imidazole group to the amidic nitrogen of the substrate. In addition, we find that the simplest model does not provide accurate results compared to the full protein system. This can be attributed to the electronic stabilization conferred by the residues around the reaction site. Interestingly, the energy profile obtained with the second scale model with additional residues shows the same trends as the full system and could therefore be considered an appropriate model system. It could be used for studying the peptide bond rupture mechanism in case full QM/MM calculations cannot be performed, or as a rapid tool for screening purposes.