Extended Data Fig. 4: Kinetic characterisation of buffer-catalysed reaction, intermediate DA variants, and DA7.

a, Non-enzymatic background reaction between Diels-Alder substrates 1 and 2 in buffer. The rates were determined in 20 mM MOPS, pH 8, 3.5% DMSO, 10 µM Zn(II), 1 mg/mL BSA at 25 °C. Error bars denote s.d. b–d, Michaelis-Menten plots for the reaction between 1 and 2 catalysed by the parental scaffold DA0 and evolutionary intermediates DA1 and DA4. An analogous plot for DA7 is shown in Fig. 2c in the main text. For DA0 (b), error bars indicate the standard deviation between three biological replicates. e, Relative activities of DA7 and the variants DA7^W16S, DA7^C35H and DA7^Q80A, which were respectively prepared to aid crystallisation and probe mechanism. Mutation of the metal binding site (DA7^{C35A/H61A/H65A}) or removal of zinc with EDTA leads to complete loss of activity. k_obs values were determined using 30 µM 1 and 60 µM 2, where [1] and [2] « K_M,1 and K_M,2 and thus (v₀/[E]₀)/([1]₀[2]₀) ≈ k_cat/(K_M,1K_M,2). f, Total turnovers for DA7 were determined using 200 µM 1 and 400 µM 2 and 10 nM enzyme. The consumption of azachalcone was corrected for contributions from the background reaction.