Activity of Free Mutants Sample Clauses

Activity of Free Mutants. The lyophilized protein stocks were rehydrated with 1.42 mg/mL TCEP solution. The protein was diluted to 1 M for the assay. 2.5 mM of DHF was prepared in a sealed xxxxx xxxx and was put through a freeze, pump, thaw cycle 3 times to remove the excess oxygen in the vial, since DHF is easily oxidized. 5 mM of NADPH was prepared in a black Eppendorf tube because NADPH is light sensitive. NAPDH and DHF using molar absorptivity of 6220 cm-1 M-1 at 340 nm and 28,000 cm-1 M-1 at 282 nm.29,31 Before the reaction occurs, 10 L of the 5 mM NADPH, 10 L of the free protein and 960 L of 50 mM sodium phosphate buffer was added to the cuvette and allowed to equilibrate for at 37C in the Ocean Optics QPOD temperature controlled stage for 3 minutes. 20 L of 50mM of DHF was added to the reaction mixture to initiate the reaction, and the reaction was monitored with an Ocean Optics QE65000 spectrometer via light from a Xenon lamp for 5 minutes. The absorbance at 340 nm was analyzed since NADPH depletion can be measured at this wavelength to determine the turnovers of the samples. A Xxxxxx Xxxxx Lambda 35 UV/Vis Spectrometer was used to determine the exact concentrations of the free protein to account for pipetting error that could have slightly altered the concentration of the free protein samples throughout prep The turnovers were determined by adjusting for NAPDH and DHF using the integrated molar absorptivity of 11,800 cm-1 M-1 at 340 nm.

Activity of Free Mutants. Activity was measured using the same methods, but at the samples were run at 6 different temperatures: 22X, 00X, 00X, 00X, 00C and 47C. Using the turnovers at the different temperatures, Arrhenius plots were created by graphing ln(turnover) vs 1/T (in Xxxxxx). Using the Arrhenius equation, the activation energies of the free proteins were determined. The equation used was = − , which was linearized to = − (1) + , where k represents the rate constant, Ea represents the activation energy, R represents the gas constant, T represents temperature in Xxxxxx and A represents the frequency factor.32 The slope of the aforementioned graph was determined to represent -Ea/R.

Activity of Free Mutants. The activity of the free mutants is important to determine that the mutations did not severely affect the activity of the enzymes. The FG Loop, Alpha Helix, Distal Active Site and His-Tag Mutant all had comparable activities to that of the WT DHFR (Table 2.1). The slightly lower turnover of the FG loop mutant may be due to FG loop motion being affected during catalysis, considering the FG Loop is one of the loops known to have the greatest motions during catalysis. Further, the E120C mutation on the FG Loop mutant is next to G121, which is known to be central to the enzyme’s catalysis, which might affect catalysis.