Fig. 3. Kinetic analysis of POP_X/Y variants in response to divalent metals.

a, b The activity of POP_X/Y variants from CFPS was assessed on Z-Ala-Pro-pNA. a Reaction rates (AU/min) at increasing Ni(II) concentrations (0–100 µM). The fold-changes between 0 and 10 µM of Ni(II) for POP_167/517, POP_167/513, POP_169/512, POP_169/510, and POP_159/517 are noted. b The reversibility of metal-dependent activity upon addition of EDTA shows near-quantitative recovery for Ni(II), Cu(II), Co(II), and Zn(II), while activity is not recovered for changes by Fe(II), Select variants from Fig. 3a are shown. c Steady-state kinetic assays performed at 85 °C using purified enzymes (~20 nM) in the presence of either 1 mM EDTA (black) or 5 µM NiCl₂ (green). Initial rates (µM/sec) are plotted versus substrate concentration (mM), and data was fit, when appropriate, with the Michaelis–Menten equation. The fold-change between reaction rate at 1 mM substrate is shown. Each data point represents the average of 3 replicates and error bars represent standard deviations. Source data are provided as a Source Data file.