Fig. 4: Ancestral versus modern catalysis by family 1 glycosidases.

a Michaelis plots of rate versus substrate concentration at pH 7 and 25 °C for hydrolysis of 4-nitrophenyl-β-D-glucopyranoside (upper panel) and 4-nitrophenyl-β-D-galactopyranoside (lower panel) catalyzed by the ancestral glycosidase with and without heme bound. v/[E]₀ stands for the rate over the total enzyme concentration. The lines are the best fits of the Michaelis–Menten equation. The different symbols (diamond, square, circle) refer to the triplicate experiments (involving two different protein preparations) performed for each protein/substrate combination. Michaelis plots for the four modern proteins studied in this work can be found in Figs. S6–S9. The values for the catalytic parameters derived from these fits are collected in Tables S2 and S3. b Logarithm of the Michaelis-Menten catalytic parameters for a glucopyranoside substrate versus a galactopyranoside substrate. pNP-glu and pNP-gal stand, respectively, for 4-nitrophenyl-β-D-glucopyranoside and 4-nitrophenyl-β-D-galactopyranoside. k_cat, K_M, and k_cat/K_M stand for the turnover number, the Michaelis constant and the catalytic efficiency. The values shown are averages of the values derived from the triplicates and the associated errors are the corresponding standard deviations. Note that, in most cases, the associated errors are smaller than the size of the data points.