Impact of mutation on proton transfer reactions in ketosteroid isomerase: insights from molecular dynamics simulations

Abstract

The two proton transfer reactions catalyzed by ketosteroid isomerase (KSI) involve a dienolate intermediate stabilized by hydrogen bonds with Tyr14 and Asp99. Molecular dynamics simulations based on an empirical valence bond model are used to examine the impact of mutating these residues on the hydrogen-bonding patterns, conformational changes, and van der Waals and electrostatic interactions during the proton transfer reactions. While the rate constants for the two proton transfer steps are similar for wild-type (WT) KSI, the simulations suggest that the rate constant for the first proton transfer step is smaller in the mutants due to the significantly higher free energy of the dienolate intermediate relative to the reactant. The calculated rate constants for the mutants D99L, Y14F, and Y14F/D99L relative to WT KSI are qualitatively consistent with the kinetic experiments indicating a significant reduction in the catalytic rates along the series of mutants. In the simulations, WT KSI retained two hydrogen-bonding interactions between the substrate and the active site, while the mutants typically retained only one hydrogen-bonding interaction. A new hydrogen-bonding interaction between the substrate and Tyr55 was observed in the double mutant, leading to the prediction that mutation of Tyr55 will have a greater impact on the proton transfer rate constants for the double mutant than for WT KSI. The electrostatic stabilization of the dienolate intermediate relative to the reactant was greater for WT KSI than for the mutants, providing a qualitative explanation for the significantly reduced rates of the mutants. The active site exhibited restricted motion during the proton transfer reactions, but small conformational changes occurred to facilitate the proton transfer reactions by strengthening the hydrogen-bonding interactions and by bringing the proton donor and acceptor closer to each other with the proper orientation for proton transfer. Thus, these calculations suggest that KSI forms a preorganized active site but that the structure of this preorganized active site is altered upon mutation. Moreover, small conformational changes due to stochastic thermal motions are required within this preorganized active site to facilitate the proton transfer reactions.

Figure 1

Schematic depiction of the proton transfer reactions catalyzed by KSI. In the first step, the proton transfers from the C4 atom of the substrate to the Asp38 residue. In the second step, the proton transfers from the Asp38 residue to the C6 atom of the substrate. The reactant, intermediate, and product states of the overall reaction are labeled.

Figure 2

Potential of mean force (PMF) curves for (a) the first proton transfer step and (b) the second proton transfer step catalyzed by KSI. These profiles are depicted for the WT (red), D99L mutant (black), Y14F mutant (blue), and Y14F/D99L double mutant (green) forms of KSI. For both steps, all curves are shifted so that the reactant is at zero energy, although mechanistically the product of the first step is the same as the reactant of the second step, following rotation of the Asp38 carboxylate group.

Figure 3

Schematic depiction of the hydrogen-bonding interactions analyzed in Figure 4.

Figure 4

Thermally averaged distances calculated along the collective reaction coordinate for the first (left) and second (right) proton transfer reactions catalyzed by KSI. Snapshots of the hydrogen-bonding pattern in the active site for the Intermediate state are also depicted. The results for the WT, D99L, Y14F, and Y14F/D99L mutants are given from top to bottom. The thermally averaged hydrogen bond donor-acceptor distances between the substrate O3 atom and Tyr14 (blue), between the substrate O3 atom and Asp99 (red), between the substrate O3 atom and Tyr55 (green), and between Tyr14 and Tyr55 (black) are shown. The distances are not shown for the mutated residue. The color coding is depicted in Figure 3.