Fig. 5: Extrinsic hydrolytic activity of the designer tri-zinc enzyme.
a Benchmark hydrolysis reaction used in this study. b Comparison of the initial reaction rates of pNA hydrolysis catalyzed by wtMIF, the MIF(S61H/Y100H) variant, and the MIF(Y100H) variant in the absence and in the presence of ZnCl2. Reaction conditions: 50 μM MIF (trimer), 150 μM ZnCl2, 2 mM pNA in 50 mM MOPS buffer (pH = 7.9) at 25 °C. c Hydrolytic activity of the MIF(S61H/Y100H) variant (orange) and the MIF(Y100H) variant (blue) as a function of the concentration of Zn2+. Reaction conditions: 50 μM MIF (trimer), 0–400 μM ZnCl2, 2 mM pNA in 50 mM MOPS buffer (pH = 7.9) at 25 °C. The black solid line represents the theoretical regression (for details, see the section ‘Estimation of the kinetic parameters’ in the Supporting Information). d, e Michaelis–Menten plots of the hydrolytic activity of wtMIF and the MIF(Y100H) variant in the absence and presence of ZnCl2. Reaction conditions: 50 μM MIF (trimer), 0 or 400 μM ZnCl2, 0.2–8 mM pNA in 50 mM MOPS buffer (pH = 7.9) at 25 °C. The blue lines in (d) represent the regression curve of the Michaelis-Menten equation. The solid and dashed lines in (e) represent the linear regression curve based on the approximation of the Michaelis-Menten equation at low substrate concentrations. In this figure, colored bar-plots and dot-plots represent mean values from three independent experiments, whilst the corresponding individual data points are displayed as black dots.
