Fig. 3: Structural, energetic and dynamic contributions to the improved catalytic rate of Des27.7.
From: Complete computational design of high-efficiency Kemp elimination enzymes
a, Mutations (grey background) at positions 136, 216 and 236 trend with increasing catalytic efficiency, which also trends with Rosetta-computed vdW energy of the catalytic Asp162. Spearman ρ = −0.88, P = 6 × 10−5. b, Analysis of the structural basis of increased KE activity by comparing substrate-bound models of Des27 (left) and Des27.7 (right). c, Percentage of MD simulation time in which the substrate is within the active site for Des27 and Des27.7 (less than or equal to 4 Å between the substrate and the active site centre of mass; blue) or distant from the active site (wheat). For 24% of the time spent in the bound conformation the substrate adopts a reactive donor-acceptor geometry (highlighted arc). The bars on the right of each pie chart show the distribution of conformations (in, out and other) in the reactive mode. d, In MD simulations, 5-nitrobenzisoxazole (sticks) can assume two catalytically competent conformations: one in which the nitro group is buried inside the TIM barrel (in, blue) and another in which it is solvent exposed (out, orange). Shown are two representative conformations from the MD simulations. e, Activation free energy for the proton abstraction of 5-nitrobenzisoxazole, comparing ΔG‡ computed from the experimentally determined kcat values (mean ± s.d. of 2 and 5 biological replicates for Des27 and Des27.7, respectively) using the Eyring rate equation, assuming T = 298 K (experiment), and the corresponding values calculated for ‘in’ and ‘out’ substrate conformations (mean ± s.d. over 30 independent EVB trajectories per system). A two-sample Wilcoxon rank-sum test (two-sided) indicated statistically significant differences in the calculated activation free energies between the ‘in’ and ‘out’ conformations for Des27.7 (P = 7.7 × 10−9) but not for Des27 (P = 0.088). R.e.u., Rosetta energy units.
