2.2 Initial rate studies
Initial reaction rates were obtained in duplicates at 30°C in 50 mM MES buffer, pH 7.0. The protein concentration was 1 - 3 µg ml-1. One substrate concentration (donor, glycerol) was varied, the other was constant at the desired/possible degree of saturation. When analyzing hydrolysis only, no glycerol was present. Samples, typically 5, were taken between 5 and 40 min. Samples were immediately quenched in the same volume of reaction buffer pre-heated to 99°C and further boiled for 10 min in a water bath.
The glucose released was measured based on a hexokinase/glucose 6-phosphate dehydrogenase assay (Goedl et al., 2007; Klotzsch & Bergmeyer, 1965). The fructose released was determined with essentially the same assay, adding phospho-glucose isomerase (Klotzsch & Bergmeyer, 1965). Detailed protocols for glucose and fructose determination are summarized in the supplementary materials and methods. Phosphate was analyzed with a colorimetric assay (Goedl et al., 2007).
Initial rates were determined from linear time courses of product release. Kinetic parameters were obtained from non-linear least-square fits (Sigma Plot 10.0) of the data with Eq. 1 – 4. Note: the equations used are phenomenological and had the purpose of fitting a single set of data. Equations derived from mechanistic models are shown in the Supporting Information Table S1. Important test for quality and consistency of the mechanistic models was to assess their ability to reproduce the results of “phenomenological fits”. In Eq. 1 – 4,\(v_{X}\) (s-1), \(v_{\text{GG}}\)(s-1) and \(v_{H}\) (s-1) are the initial rates of the donor substrate consumption measured as release of the leaving group X (= fructose or phosphate), GG release and hydrolysis, respectively. \(v_{H}\) was recorded in the presence and absence of glycerol. Note: \(v_{X}\) is the sum of \(v_{\text{GG}}\) and\(v_{H}\). [GlcX] and [GOH] are the donor and glycerol concentration, respectively. Throughout this work, initial rates were obtained from volumetric rates [µmol ml-1s-1] divided by molar enzyme concentration. The molar concentration is determined from the protein concentration, using a molecular mass of 55 kDa for Lm SucP (Goedl et al., 2007) and 56 kDa for Ba SucP (Sprogøe et al., 2004).\({}^{\text{app}}V_{X}\) is an apparent maximum rate constant and\({}^{\text{app}}K\) an apparent Michaelis constant, while\({}^{\text{app}}K_{i}\) is an apparent substrate inhibition constant The transfer coefficient (TC) indicates the rate ratio’s (\(v_{\text{GG}}/v_{H}\)) dependency on [GOH].