Kinetic data from different acetoacetyl-CoA reductases
These folders contain experimental records, calculation datasheets and scripts to simulate or find best-fitted kinetic parameters from certain enzymatically catalyzed reactions. These reactions were catalyzed by four engineered enzymes derived from the acetoacetyl-CoA reductase encoded by the phaB1 gene from Cupriavidus necator. The DNA and amino acid sequences of these enzymes are provided. It is also provided the DNA sequence map of the vector where the amino acid encoding sequences for these engineered enzymes were inserted. Inside the folder named "kinetic data" it is also possible to find a TXT file with a detailed description of the content. The experimental conditions of the enzymatic assays is carefully explained. Briefly, this folder contains (1) the raw experimental data recorded with the software Gen 5 (Biotek). (2) It is also possible to find Microsoft Excel datasheets with the records of raw absorbance in time together with initial substrates, initial enzyme and product concentrations in time (reaction progress curves). Most of these progress curves were analyzed with the software DYNAFIT (Biokin) to obtain the kinetic parameters. The progress curves highlighted in red in the Microsoft Excel files were not considered for the statistical analysis due to experimental errors. The progress curves highlighted in yellow in the Microsoft Excel files were considered after the elimination of some outlier points. (3) Scripts enabling two kind of statistical analyses with DYNAFIT were included: scripts to make model discrimination analyses of the different reaction progress curves obtained in every single experiment, and scripts to calculate the confidence intervals for the kinetic parameters using a Monte Carlo approach. We included the scripting manual of DYNAFIT to understand the synthaxis of these scripts .(4) It is included two MATLAB scripts to make a comparison between the kinetic parameters obtained using the Michaelis-Menten model and the true kinetic parameters of a BiBi reversible reaction. (5) Finally, we included DYNAFIT, MATLAB and Microsoft Excel files enabling the calculation of the relative use of NADH over NADPH by one of the engineered enzymes, and also to calculate the metabolic flux capacity of this enzyme.
Steps to reproduce
Substrates stocks were always freshly prepared from reagents with analytical grade (purchased from Santa Cruz Biotechnology or Sigma-Aldrich). The substrate concentrations in such stocks were estimated by spectrophotometry, using the Lambert-Beer law. Acetoacetyl-CoA (AcAcCoA) stocks were prepared in distilled water (slightly acid pH favors the stability of AcAcCoA solutions). However, the substrate concentrations in these freshly prepared AcAcCoA stocks were assessed as follows: a solution (at a known dilution) of the freshly prepared stock was prepared in W buffer ((W buffer: Tris-HCl 50 mM, NaCl 5 mM, MgCl2 5 mM, pH 8.0)). The absorbance of this solution, at 310 nm, was recorded, using W buffer as the blank. The use of W buffer ensured stable and known H+ and Mg2+ concentrations (the extinction coefficient of AcAcCoA steeply change with small changes in Mg2+ and H+ concentrations, showing an ε310nm = 11000 M-1cm-1 at pH 8.0 and Mg2+ 5 mM [Stern, J. R. (1956) Optical properties of aceto-acetyl-S-coenzyme A and its metal chelates, J Biol Chem. 221, 33-44.]). In the case of NAD(P)H, the stocks were prepared and quantified in MOPS buffer (((3-(N-morpholino)-propanesulfonic acid) 50 mM, NaCl 5 mM, MgCl2 5 mM, pH 7.0)). The reference molar extinction coefficient of ε340nm = 6220 M-1cm-1 was employed to evaluate the concentrations of the NAD(P)H stocks. Kinetic assays were performed in MOPS buffer, at 30 Celsius, in a Synergy HTX plate reader (Biotek), using 96 wells half-area microplates (Greiner, code 675101). Changes in substrate concentrations were followed by spectrophotometry, observing the changes in absorbance at 340 nm or 360 nm. To determine the apparent extinction coefficients to be used at 360 nm, some dilutions, at known dilution factor of AcAcCoA and NAD(P)H in MOPS buffer, were prepared. The absorbance values were measured and with these experimental values, apparent extinction coefficients for AcAcCoA (23 M-1cm-1) and NAD(P)H (4075 M-1cm-1) at 360 nm were determined. With also present a kinetic model to calculate the NAD(P)H consumption rates. This kinetic model was constructed based on the following assumptions: (i) the free enzyme interacts with AcAcCoA, forming an E-AcAcCoA complex. (ii) The E-AcAcCoA complex can interact either with NADH or NADPH. (iii) An additional AcAcCoA molecule can interact either with the E-AcAcCoA-NADH complex or the E-AcAcCoA-NADPH complex, forming the E-(AcAcCoA)2-NADH complex or the E-(AcAcCoA)2-NADPH complex, respectively. (iv) All the intermolecular interactions, except the chemical steps where the reaction products are generated, are considered in rapid-equilibria. These equilibria are characterized by the corresponding KM or KiS. (v) The reaction products are formed in first-order irreversible processes.