Fig. 4: Kinetic solvent viscosity effects.

a Kemp elimination reaction scheme. b–e Gray and black lines correspond to Core and Evolved variants, respectively. Kinetic solvent viscosity effects on k_cat/K_M for b HG3, c 1A53, and d KE70 variants show a linear dependence on relative viscosity (η_rel). The slope (m) corresponds to k₃/(k₂ + k₃), where k₂ is the rate constant of substrate dissociation and k₃ is the rate constant for the chemical transformation. A slope of 1 indicates that k₃ is at least 100-fold larger than k₂ (m = k₃/k₃). A slope of 0 indicates that k₃ is much smaller than k₂ (m = k₃/k₂ = 0). e Kinetic solvent viscosity effects on k_cat for KE70-Evolved show an inverse hyperbolic pattern, which is consistent with the presence of a solvent-sensitive internal isomerization of the enzyme-product complex. Data represent the average of 6 individual replicates from 2 independent protein batches, with error bars reporting the SEM. Source data are provided as a Source data file.