Figure 2
Spectrophotometric analysis of phosphoethanolamine methyltransferase activity. (A) PfPMT-catalyzed methylation of P-EA. Reaction mixtures contained 200 μM SAM, 200 μM P-EA, 1000 μM MnSO4, 0.5 μM BsAda, 4.72 μM SAHN and 0 μM (red) or 2.5 μM (blue) of purified PfPMT enzyme in 100 mM HEPES assay buffer pH 7.5. The decrease in absorbance was monitored at 265 nm. The reactions components were kept the same as stated above in panels B-E, except when noted. (B) Dependence of the rate of PfPMT-catalyzed reaction on different concentrations of the coupling enzymes. Reactions contained BsAda 0.25 μM and SAHN 2.13 μM (orange), 0.5 μM BsAda, 4.72 μM SAHN (blue) and without PfPMT (red), and BsAda 1 μM and SAHN 8.5 μM (green). (C) Dependence of the rate of PfPMT-catalyzed reaction on different concentrations of SAM and P-EA. Reactions contained P-EA 50 μM and SAM 150 μM (orange), P-EA 100 μM and SAM 100 μM (black), p-Etn 150 μM and SAM 150 μM (green), P-EA 200 μM and SAM 200 μM (blue), and without PfPMT (red). (D) Effect of PfPMT concentration on its activity. Reactions contained 0 μM PfPMT (red), 312.5 nM PfPMT (orange), 625 nM PfPMT (green), 1.25 μM PfPMT (black), and 2.5 μM (blue). (E) Effect of the pH and buffer composition on PfPMT activity. Reaction mixtures contained 200 μM SAM, 200 μM P-EA, 1000 μM MnSO4, 0.5 μM BsAda, 4.72 μM SAHN and 0 μM (red) or 2.5 μM (black), of purified PfPMT enzyme in 100 mM HEPES assay buffer pH 7.5. Reactions also contained 100 mM HEPES assay buffer pH 7 (green), and pH 8 (cyan), as well as 100 mM Tris-HCl assay buffer pH 7 (orange), pH 7.5 (purple), and pH 8 (yellow). Results are representative of three independent experiments.