Sandbox Reserved 770: Difference between revisions
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Most of the conserved active site residues are contained within seven stabilizing alpha helices, represented by Figure 6. These residues include: Leu266, Asn270, Val269, Leu215, Lys486, and Ile472.<ref name=rutgers>http://maptest.rutgers.edu/drupal/?q=node/408</ref> Six positive alpha helices point toward the active site in association with MIO cofactor. This association will not only increase the electrophilicity of MIO, but also increases he positive charge of highly conserved Lys468 residue. The positive poles are suitable for stabilizing a carbanionic charge produced by an elimination unimolecular conjugate base (E1cB) mechanism of substrate phenylalanine, in accordance with MIO cofactor. Stabilization of the carbanion reduces pKa of the C3 methylidine group of phenylalanine, promoting the interactions between the negatively charged carboxylate end of phenylalanine to the active site of PAL. | Most of the conserved active site residues are contained within seven stabilizing alpha helices, represented by Figure 6. These residues include: Leu266, Asn270, Val269, Leu215, Lys486, and Ile472.<ref name=rutgers>http://maptest.rutgers.edu/drupal/?q=node/408</ref> Six positive alpha helices point toward the active site in association with MIO cofactor. This association will not only increase the electrophilicity of MIO, but also increases he positive charge of highly conserved Lys468 residue. The positive poles are suitable for stabilizing a carbanionic charge produced by an elimination unimolecular conjugate base (E1cB) mechanism of substrate phenylalanine, in accordance with MIO cofactor. Stabilization of the carbanion reduces pKa of the C3 methylidine group of phenylalanine, promoting the interactions between the negatively charged carboxylate end of phenylalanine to the active site of PAL. | ||
==Conserved Residues in Catalysis== | ==Conserved Residues in Catalysis Mechanism== | ||
[[Image:PAL_CIN_Interactions.png|right|thumb|Figure 8. Lysine468 attachment to carboxyl group of substrate CIN [[Ligand]]]] | [[Image:PAL_CIN_Interactions.png|right|thumb|Figure 8. Lysine468 attachment to carboxyl group of substrate CIN [[Ligand]]]] | ||
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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== | ||
