Sandbox Reserved 770: Difference between revisions
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'''Histidine137''' | '''Histidine137''' | ||
Formation of carbanion intermediate is promoted by the positioning of the NH2 group of Asn270 to share a hydrogen bond with the enolate oxygen of MIO, increasing the electropositivity of MIO methylidene group, shown in Figure 9. Abstraction of the pro-''S'' hydrogen from C3 of substrate to form product ''trans''-cinnamic acid is catalyzed by His137 residue on helix one, which is connected to loop regions on both its C and N termini, allowing for its movement. His137 is located near the C terminus with its negative pole directed toward the active site, thus increasing basicity of His137 so it can act as a base. Development and further stabilization of the carbanion are provided by the phenyl group of the substrate, helix dipoles directing positive poles toward residues, improved MIO electron-withdrawing capability (enhanced by positive poles of three alpha helices), and electron withdrawal from substrates carboxy group by residues of positive pole N termini in alpha helices. MIO group assists in the breakage of C-N bond of substrate to proceed carbanion intermediate to product ''trans''-cinnamate. Once C-N bond is broken by PAL, cinnamate leaves the active site by Lys468 chaperone. | Formation of carbanion intermediate is promoted by the positioning of the NH2 group of Asn270 to share a hydrogen bond with the enolate oxygen of MIO, increasing the electropositivity of MIO methylidene group, shown in Figure 9. Abstraction of the pro-''S'' hydrogen from C3 of substrate to form product ''trans''-cinnamic acid is catalyzed by His137 residue on helix one, which is connected to loop regions on both its C and N termini, allowing for its movement. His137 is located near the C terminus with its negative pole directed toward the active site, thus increasing basicity of His137 so it can act as a base. Development and further stabilization of the carbanion are provided by the phenyl group of the substrate, helix dipoles directing positive poles toward residues, improved MIO electron-withdrawing capability (enhanced by positive poles of three alpha helices), and electron withdrawal from substrates carboxy group by residues of positive pole N termini in alpha helices. MIO group assists in the breakage of C-N bond of substrate to proceed carbanion intermediate to product ''trans''-cinnamate. Once C-N bond is broken by PAL, cinnamate leaves the active site by Lys468 chaperone. | ||
==Methods Used to Solve Structure== | ==Methods Used to Solve Structure== | ||
As Described in Crystal Structure of Phenylalanine Ammonia Lyase by Calabrese et al. <ref name="crystal">http://pubs.acs.org.prox.lib.ncsu.edu/doi/pdfplus/10.1021/bi049053%2B</ref> | |||
'''A) Expression and Purification'''. Escherichia coli cells containing plasmid pEAL (PAL inserted into pET-24a) were grown in media methionine | |||
'''B) Protein Isolation by Centrifugation''' | |||
'''C) Protein was purified by three chromatography steps''': | |||
(1) Anion-exchange chromatography | |||
(2) Hydrophobic interaction chromatography | |||
(3) Gel-filtration chromatography | |||
'''D) Protein Concentrated''': by use of a Centricon system (Amersham Biosystems). | |||
'''E) SDS−PAGE analysis''': of the preparation, detection of protein and contaminants | |||
'''F) Crystallizations''': Hanging-drop vapor diffusion method | |||
'''G) Molecular Replacement''': The monoclinic structure was solved via use of the program MOLREP | |||
'''Electron Density aps''': Using [X-Ray Diffraction] were displayed and models were manually constructed with the programs O (33) and XtalView/Xfit | |||
'''Refinement Cycles''': performed with the program CNX, coordinates for the MIO moiety initially taken directly from the HAL structure | |||
==References== | ==References== | ||
<references /> | <references /> | ||
