Tored by SDS-PAGE and Bradford assay, respectively.excitation wavelength of 290 nm was used. Emission spectra were recorded from 300 to 420 nm, using a spectral slit width of 2 nm for the excitation and 3 nm for the emission monochromator. To minimize Trp photobleaching, the spectra were acquired using a fast scanning mode (2.5 nm per step, 0.5 s integration time). Before measurements, all samples were carefully temperatureequilibrated. Enzyme concentration was 0.1 mg/mL (2.7 nmol of monomers/mL) in 50 mM MOPS buffer, pH 7.0, 37uC. Conditions under which specific spectra were recorded are provided in the text, tables, and figure legends. Control experiments demonstrated that, if several spectra of FBPase were taken without any additions, they were completely superimposed. All kinetic experiments were performed at pH 7.0 and 37uC using a glucose-6-phosphate isomerase glucose-6-phosphate dehydrogenase coupled spectrophotometric assay [27] and 50 mM MOPS buffer, pH 7.MOG peptide (35-55) 0, 37uC. The forward FBPase reaction was started with the saturating concentration of F1,6P2 (50 mM).Deucravacitinib One unit of enzyme activity is defined as the amount of the enzyme that catalyzes the formation of 1 mmol of product per minute. The reverse FBPase reaction was measured in a mixture containing: 50 mM MOPS, 150 mM KCl, 2.25 mM MgCl2, 0.25 mM EDTA, 5 mM fructose-6-phosphate, 5 mM KPi; 0.1 mM NADH, 5 U/mL of rabbit muscle aldolase, 10 U/mL of triose-3-phosphate isomerase and 10 U/mL of glycerol-3phosphate dehydrogenase, pH 7.0, 37uC. Spectrophotometric measurements were performed with the Agilent 8453 diode array spectrophotometer. Determination of kinetic parameters such as the dissociation constant of the enzymesubstrate complex (Ks), the inhibition constant of FBPase by its substrate (Kis), b and the catalytic rate constant (kcat) were performed assuming the model of partial non-competitive inhibition by substrate, which assumes that F1,6P2 may associate with the canonical active site and the inhibitory site, which also catalyses the hydrolysis of the substrate but the kcat is lower [27]. The overall velocity at which product is formed may be written as followed: v Vmax (1zb =Kis )=(1zKs = zKs =Kis z =Kis ) Where: Ks is an enzyme-substrate dissociation constant, Kis is the inhibition constant of FBPase by substrate and b is the ratio of kcat when substrate binds to the inhibitory site to kcat when substrate binds only to the active site.PMID:24278086 The values of Ki and n for AMP and Ka and n for Mg2+ were calculated using the Hill equation [28]. The effect of Ca2+ on the activation of FBPase by Mg2+ was analyzed using the Michaelis enten kinetics-derived equation describing competitive inhibition (Fig. 1 C) [28]. In brief, the effect of competitive inhibition by Ca2+, in respect to Mg2+, may be written as (2): v0 Vmax Mg2z = KaMg2z1z Ca2z =KiCa2z z Mg2zSteady-state Fluorescence and Enzyme Kinetic MeasurementsFluorescence data were collected using a Fluorolog 3 (SpexHoriba) fluorometer. To avoid exciting tyrosyl side chains, anPLOS ONE | www.plosone.orgwhere: v0 is reaction velocity, Vmax is the maximal velocity, [Ca2+] is the concentration of the inhibitor (Ca2+), [Mg2+] is Mg2+ concentration, and Ka Mg2+ is the dissociation constant for Mg2+ determined in the absence of the inhibitor.Ca2+ Competes with Mg2+ for Binding to FBPaseFigure 1. The effect of Ca2+ on kinetic parameters of wild-type and mutated form of muscle FBPase. A) Activation of the Tyr57Trp muscle FBPase mutant by Mg2+.
