Proton Network Flexibility Enables Robustness and Large Electric Fields in the Ketosteroid Isomerase Active Site

Abstract

Hydrogen-bond networks play vital roles in biological functions ranging from protein folding to enzyme catalysis. Here we combine electronic structure calculations and ab initio path integral molecular dynamics simulations, which incorporate both nuclear and electronic quantum effects, to show why the network of short hydrogen bonds in the active site of ketosteroid isomerase is remarkably robust to mutations along the network and how this gives rise to large local electric fields. We demonstrate that these properties arise from the network's ability to respond to a perturbation by shifting proton positions and redistributing electronic charge density. This flexibility leads to small changes in properties such as the partial ionization of residues and pK isotope effects upon mutation of the residues, consistent with recent experiments. This proton flexibility is further enhanced when an extended hydrogen-bond network forms in the presence of an intermediate analogue, which allows us to explain the chemical origins of the large electric fields in the enzyme's active site observed in recent experiments.

Computational optimization of electric fields for better catalysis design

“…8 We found that the scaffold plays a stabilizing role for the KSI catalytic activity, contributing +16 MV/cm (~28%) to the total electric field, although an additional +41 MV/cm emanated from the local active site environment, thereby contributing a majority share. 8 Markland and co-workers confirmed that the scaffold exerts an overall stabilizing electric field from ab initio molecular dynamics (AIMD) simulations that include Nuclear Quantum Effects (NQE) 16 13,16 ; this is because the electric field calculated in the reactant and transition states are different and cannot be factorized in front of the reaction dipole difference. 8,22 Starting from the original design with a kcat/KM of 27 M -1 s -1 , we created a computationally improved variant through introduction of 4 targeted mutations to yield a kcat/KM of 403 M -1 s -1 .…”

“…68 Although earlier approximations have been used by Warshel and co-workers that take into account NQEs in enzyme catalysis 69 , recent theoretical work have successfully focused on significant cost reduction of NQEs 70 or increasing their accuracy through electronic-nuclear coupling 71 . While the quantum nature of the nuclei have been shown to be important in water and enzyme active sites 16 , they are still typically ignored in the calculation of the electric field and their effects on the ground state and transition states of the chemical step. This may change in the future as algorithmic improvements for calculating NQEs become more tractable 70 , and their importance for enzymes 16 and nanocatalysts 16,68 continues to be established.…”

“…While the quantum nature of the nuclei have been shown to be important in water and enzyme active sites 16 , they are still typically ignored in the calculation of the electric field and their effects on the ground state and transition states of the chemical step. This may change in the future as algorithmic improvements for calculating NQEs become more tractable 70 , and their importance for enzymes 16 and nanocatalysts 16,68 continues to be established.…”

Fluctuations of Electric Fields in the Active Site of the Enzyme Ketosteroid Isomerase

“…and Asp-103. 8 Previous theoretical studies have emphasized the need for polarization to compute electric fields 9,21 ; to further illustrate this point, our simulations ran without mutual polarization reduces the average electric field by 40 MV/cm (-108.93 ± -4.9 to -68.08 ± -3.1). Hence our calculations using full mutual polarization can capture nearly all the physics governing the production of electric fields in KSI, for which it has the undeniable advantage of accessing longer timescales relevant to this study.…”

“…7 For KSI much work has been devoted to the energetics of the active site and in particular to the role of the extended hydrogen bond network that stabilizes the oxyanion hole. 8 A previous study found compelling evidence for long-range structural coupling within the hydrogen bond network of a group of tyrosines, with minimal influence of the remainder of the protein scaffold. 9…”