Fig. 3: Steady-state and transient kinetic analysis of CTZ conversion.
From: Engineering the protein dynamics of an ancestral luciferase
The kinetic models consist of an enzyme E, a substrate S, an enzyme-substrate complex in two conformations (E.S and E*.S), an enzyme-product complex E.P, and a product P. a Steady-state kinetic parameters (Michaelis constant Km, turnover number kcat, enzyme-product complex dissociation constant Kp) were determined with the substrate CTZ in 100 mM phosphate buffer at pH 7.5 and 37 °C by global analysis of triplicates of full progress curves recorded with at least five concentrations of CTZ. b Results of pre-steady-state kinetic analysis of the CTZ substrate binding in 100 mM phosphate buffer at pH 7.5 and a lower temperature (15 °C). This enabled identification of the induced fit substrate binding mechanism, involving initial collision of the enzyme and the substrate (described by forward rate constant k+1 and reverse rate constant k−1), followed by a conformational change of the enzyme induced by the bound substrate (described by forward rate constant k+2 and reverse rate constant k−2). A simple binding mechanism including only the first step was observed for AncHLD-RLuc. The kinetic parameters were determined by global fitting of tryptophan fluorescence traces obtained with at least 10 concentrations of CTZ and 10 concentrations of each tested enzyme, in each case with seven replicates (Supplementary Note 6). The data are presented as best fit values ± standard errors (S.E.) calculated from the covariance matrix during nonlinear regression. Source data is available as a Source data file for Fig. 3.
