Sandbox Reserved 770: Difference between revisions
From Proteopedia
Jump to navigationJump to search
Bryan Toton (talk | contribs) |
Bryan Toton (talk | contribs) |
||
| Line 49: | Line 49: | ||
The central core within each monomer of PAL contains three central alpha helices of triple-coiled coils (Helix 3,Helix 4, and Helix 7), improving its rigidity similar to fibrous proteins in keratin. The three core helices are oriented with similarly aligned dipoles to create an electro-positive platform for cofactor 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) to anchor through noncovalent bonding, shown in Figure 4b. Covalent linkages between cofactor MIO and PAL backbone act to direct the C-terminus of MIO to an alpha helix's N-terminus, creating a loop, thus the helix's positive pole points toward the cofactor and the active site. PAL active site contains a highly conserved Ala-Ser-Gly triad. Post-translational modification of an electrophilic prostetic group MIO formed autocatalytically by cyclization and dehydration of PAL's active site. MIO provides the surface for phenylalanine conversion to produce trans-cinnamic acid and ammonia. | The central core within each monomer of PAL contains three central alpha helices of triple-coiled coils (Helix 3,Helix 4, and Helix 7), improving its rigidity similar to fibrous proteins in keratin. The three core helices are oriented with similarly aligned dipoles to create an electro-positive platform for cofactor 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) to anchor through noncovalent bonding, shown in Figure 4b. Covalent linkages between cofactor MIO and PAL backbone act to direct the C-terminus of MIO to an alpha helix's N-terminus, creating a loop, thus the helix's positive pole points toward the cofactor and the active site. PAL active site contains a highly conserved Ala-Ser-Gly triad. Post-translational modification of an electrophilic prostetic group MIO formed autocatalytically by cyclization and dehydration of PAL's active site. MIO provides the surface for phenylalanine conversion to produce trans-cinnamic acid and ammonia. | ||
Most of the conserved active site residues are contained within seven stabilizing alpha helices. 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 | Most of the conserved active site residues are contained within seven stabilizing alpha helices. 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. | ||
The positively charged side chain of Lys468 recognizes the carboxyl group of substrate by forming a salt bridge, when it is located in the mouth of the funnel. Before the side chain encloses, Lysine chaperones the substrate to its reactive position for sharing additional interactions between substrate's carboxyl group and side chains Glu496 and Gln500. Lys468 is strictly conserved with almost always adjacent Gly amino acid, which would improve mobility of Lys486 chaperone ability for substrate. Lys486 also acts to place the NH2 group of the substrate near MIO to ensure the carboxylate group of substrate does not react nonproductively with methylidene of MIO by forming an ester. | |||
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. Abstraction of the pro-''S'' hydrogen from C3 ofsubstrate 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. | |||
==References== | ==References== | ||
<references /> | <references /> | ||
