Figure 7: Various activities of the W229D/F290W variant of the GNCA4 scaffold.
From: De novo active sites for resurrected Precambrian enzymes
(a) Activities linked to the new active site. (b) Activities linked to the natural preexisting active site. Plots of rate versus substrate concentration are shown in (a,b), with the continuous lines representing the best fits based on the Michaelis–Menten equation. Ester hydrolysis data correspond to p-nitrophenyl acetate hydrolysis and were obtained with the GNCA4-W229D/F290W variant (closed data points) and also with this variant modified to block any esterase activity at the natural, antibiotic degradation site. This was achieved by mutating the catalytic S70 to alanine (open squares) or by inactivation by clavulanic acid (open triangles). Catalytic parameters for ester hydrolysis discussed in the Main Text are derived from the Michaelis–Menten fit to the modified variants. The equation used to fit to the data of the unmodified variant includes an additional linear term to account for esterase activity at the antibiotic degradation site. The esterase catalytic efficiency at the natural active site is, however, found to be ∼20-fold smaller than that at the new active site.
