| Popis: |
This work combines the use of thio effects and site-directed mutagenesis (SDM) to characterize the catalytic mechanisms of phosphatidylinositol-specific phospholipase Cs (PI-PLCs). Eukaryotic PI-PLCs utilize Ca2+ in catalysis, whereas Bacillus thuringiensis PI-PLC (btPLC) uses a spatially conserved guanidinium group. A Ca2+-dependent mutant of btPLC was constructed and characterized to understand the functional differences between these two positively charged moieties. The following results indicate that a true catalytic metal site was created by a single mutation at position 69: (1) R69D was activated by Ca2+. (2) Titration of R69D with Ca2+, monitored by 15N-1H HSQC NMR, showed that addition of Ca2+ to R69D restores the environment of the catalytic site analogous to that attained by WT. (3) Upon Ca2+ activation, the SP-thio effect (kO/kSp = 4.4 E5) approached a value similar to that of WT, suggesting a structural and functional similarity between Arg69 and Asp69-Ca2+. (4) Results from additional SDM suggest that the Ca2+ binding site is comprised of side chains from Asp33, Asp67, Asp69, and Glu117. A natural occurring metal-dependent Streptomyces antibioticus PI-PLC (saPLC1) was cloned, expressed, purified, and characterized. The results enrich the knowledge in application of thio effect and provide useful mechanistic information for metal-dependent PI-PLCs: (1) WT has an extraordinarily high SP-thio effect (kO/kSp = 1.6 E8) in the presence of Ca2+. Substitution of Ca2+ by Cd2+ in WT enhances the activity toward SP-DPPsI by a factor of 400, but does not affect the activity with PI or RP-DPPsI, which are evident that the metal cofactor interacts with the pro-SP oxygen. (2) In the presence of Ca2+, the RP-thio effect of H16A (kO/kRp = 1.1) is lowered from that of WT (kO/kRp = 47). Ca2+/Cd2+ replacement in H16A dramatically increases the RP-thio effect (kO/kRp = 7.9 E3). Tentatively, these results suggest that both Ca2+ and His16 are involved in the interaction with the pro-SP oxygen. (3) WT and most of its mutants demonstrated small bridging thio effects (kO/kS = 0.5 ~ 2), whereas H55A displays an inverse thio effect (kO/kS) of 0.0019, implicating that His55 functions as the general acid in the phosphotransferase reaction. This work combines the use of thio effects and site-directed mutagenesis (SDM) to characterize the catalytic mechanisms of phosphatidylinositol-specific phospholipase Cs (PI-PLCs). Eukaryotic PI-PLCs utilize Ca2+ in catalysis, whereas Bacillus thuringiensis PI-PLC (btPLC) uses a spatially conserved guanidinium group. A Ca2+-dependent mutant of btPLC was constructed and characterized to understand the functional differences between these two positively charged moieties. The following results indicate that a true catalytic metal site was created by a single mutation at position 69: (1) R69D was activated by Ca2+. (2) Titration of R69D with Ca2+, monitored by 15N-1H HSQC NMR, showed that addition of Ca2+ to R69D restores the environment of the catalytic site analogous to that attained by WT. (3) Upon Ca2+ activation, the SP-thio effect (kO/kSp = 4.4 E5) approached a value similar to that of WT, suggesting a structural and functional similarity between Arg69 and Asp69-Ca2+. (4) Results from additional SDM suggest that the Ca2+ binding site is comprised of side chains from Asp33, Asp67, Asp69, and Glu117. A natural occurring metal-dependent Streptomyces antibioticus PI-PLC (saPLC1) was cloned, expressed, purified, and characterized. The results enrich the knowledge in application of thio effect and provide useful mechanistic information for metal-dependent PI-PLCs: (1) WT has an extraordinarily high SP-thio effect (kO/kSp = 1.6 E8) in the presence of Ca2+. Substitution of Ca2+ by Cd2+ in WT enhances the activity toward SP-DPPsI by a factor of 400, but does not affect the activity with PI or RP-DPPsI, which are evident that the metal cofactor interacts with the pro-SP oxygen. (2) In the presence of Ca2+, the RP-thio effect of H16A (kO/kRp = 1.1) is lowered from that of WT (kO/kRp = 47). Ca2+/Cd2+ replacement in H16A dramatically increases the RP-thio effect (kO/kRp = 7.9 E3). Tentatively, these results suggest that both Ca2+ and His16 are involved in the interaction with the pro-SP oxygen. (3) WT and most of its mutants demonstrated small bridging thio effects (kO/kS = 0.5 ~ 2), whereas H55A displays an inverse thio effect (kO/kS) of 0.0019, implicating that His55 functions as the general acid in the phosphotransferase reaction. |
