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With recombinant human carboxylesterase 1; water; In aq. phosphate buffer; at 37℃; for 0.133333h;Enzymatic reaction;Kinetics; |
General procedure: Michaelis-Menten kinetic parameters were determined forTPL, RPL, and EPL in recombinant human CES1. An initial study wasperformed to establish time and protein linearity of the reactions by measuringproduct formed at several time points (5, 10, 15, 20, and 30 minutes) usingthree protein concentrations (50, 100, and 150 mg/ml). Final protein andsubstrate concentrations, as well as incubation times, were selected such thata quantifiable amount of hydrolysis product was formed and so that thesampling was done in the linear range of the reactions. No more than 20% ofsubstrate was hydrolyzed at the time of sampling. Substrate concentrationswere 0.2-4 mM for TPL, 0.1-4 mM for RPL, and 0.05-4 mM for EPL. Finalrecombinant CES1 protein concentrations were 50 mg/ml for TPL and RPL,and 100 mg/ml for EPL. No organic solvent was present in the incubations. Thedrug and enzyme combinations were incubated in 96-well, twin.tec polymerasechain reaction plates (Eppendorf, Hamburg, Germany) in 100 mM phosphatebuffer at 37C, in a final reaction volume of 50 ml. Control incubations with noenzyme were performed for each compound. Reactions (8 minutes for RPL,10 minutes for TPL, and 20 minutes for EPL) were terminated by transferring20 ml to an equal volume of cold acetonitrile with 0.5% formic acid and thecorresponding internal standard-500 nM trandolaprilat (TPLA)-d5 for TPL orRPL, and 1,000 nM enalaprilat (EPLA)-d5 for EPL. Next, 110 ml of mobilephase was added, and the samples were centrifuged at 2,000g, 5C, for10 minutes. The supernatant was analyzed by liquid chromatography-tandemmass spectrometry as described in the analytical methods section below. Thehydrolysis rate versus the substrate concentration was plotted and fit witha standard Michaelis-Menten equation or a substrate inhibition equation, wherea second substrate molecule was treated as an uncompetitive inhibitor (Cornish-Bowden, 2012). The two fits were compared with an extra-sum-of-squaresF-test. The test compares the improvement of sum-of-squares with the morecomplicated model (the substrate inhibition model) versus the loss of degrees offreedom. The null-hypothesis was that the data were adequately described bythe standard Michaelis-Menten equation. P values , 0.05 were consideredsignificant. Nonlinear regression fitting and statistical analysis was performedwith Prism, version 6.02 (GraphPad Software, Inc., San Diego, CA). Intrinsicclearance (CLint) was calculated by dividing Vmax by Km. |