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].