Sandbox Reserved 770: Difference between revisions

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[[Image:Quaternary_Structure.gif|thumb|right|Figure 3. Quaternary Structure of PAL. The four individual monomers are color-coded in red, green, blue, and yellow, displaying the approximate 222 symmetry of the PAL tetramer.(a) Stereoview of PAL from a side perspective. The bracketed areas represent the residues, arranged in a fan, that are present in PAL and absent in HAL. (b) Top perspective of the PAL tetramer. This view looks down into the active sites of two of the subunits; the other two active sites would be seen from a bottom view.]]  
[[Image:Quaternary_Structure.gif|thumb|right|Figure 3. Quaternary Structure of PAL. The four individual monomers are color-coded in red, green, blue, and yellow, displaying the approximate 222 symmetry of the PAL tetramer.(a) Stereoview of PAL from a side perspective. The bracketed areas represent the residues, arranged in a fan, that are present in PAL and absent in HAL. (b) Top perspective of the PAL tetramer. This view looks down into the active sites of two of the subunits; the other two active sites would be seen from a bottom view.]]  


Phenylalanine ammonia lyase enzyme overall structure is 54% helical (31 helices; 387 residues)and 4% beta sheet (15 strands; 32 residues). PAL is a homo-dimer composed of two identical subunits.<ref name="crystallization">http://ci.nii.ac.jp/els/110006324658.pdf?id=ART0008332067&type=pdf&lang=en&host=cinii&order_no=&ppv_type=0&lang_sw=&no=1386328093&cp=</ref> Each subunit of PAL from ''R. toruloides'' assume a "seahorse" shape by interlocking head-to-tail, creating overlapping regions with two adjacent subunits, as shown by Figure 2. These overlapping regions maximize interactions between subunits, giving rise to the formation of the tightly assembled tetramer, as shown in Figure 3. Formation of the tetramer buries 58% of their combined surfaces. <ref name=crystal>http://pubs.acs.org.prox.lib.ncsu.edu/doi/pdfplus/10.1021/bi049053%2B</ref> Of the 66 interactions between adjacent subunits, 25 hydrogen bonding interactions exists between Asp & Glu carboxylate oxygens and NH2 & OH moieties, including a prominent band of Asp & Glu interactions with Arg side chains between subunits nearby the central bundle of helices. PAL's central core is comprised of parallel alpha helices of varying lengths. There is only one section of Beta sheet longer than three residues in PAL, which resides in the funnel region leading to the active site. PAL and HAL (Histadine Ammonia Lyase) contain similar folds, but PAL differs from HAL with 215 additional residues. Of the 215 residues from this section, 155 residues extend above and below the main body of the structure, creating a "fan" arrangement, shown as the bracketed areas in Figure 3a.
Phenylalanine ammonia lyase enzyme overall structure is 54% helical (31 helices; 387 residues)and 4% beta sheet (15 strands; 32 residues).<ref name="pncbsequence">http://www.rcsb.org/pdb/explore/remediatedSequence.do?params.chainEntityStrategyStr=all&structureId=1T6J</ref> PAL is a homo-dimer composed of two identical subunits.<ref name="crystallization">http://ci.nii.ac.jp/els/110006324658.pdf?id=ART0008332067&type=pdf&lang=en&host=cinii&order_no=&ppv_type=0&lang_sw=&no=1386328093&cp=</ref> Each subunit of PAL from ''R. toruloides'' assume a "seahorse" shape by interlocking head-to-tail, creating overlapping regions with two adjacent subunits, as shown by Figure 2. These overlapping regions maximize interactions between subunits, giving rise to the formation of the tightly assembled tetramer, as shown in Figure 3. Formation of the tetramer buries 58% of their combined surfaces. <ref name=crystal>http://pubs.acs.org.prox.lib.ncsu.edu/doi/pdfplus/10.1021/bi049053%2B</ref> Of the 66 interactions between adjacent subunits, 25 hydrogen bonding interactions exists between Asp & Glu carboxylate oxygens and NH2 & OH moieties, including a prominent band of Asp & Glu interactions with Arg side chains between subunits nearby the central bundle of helices. PAL's central core is comprised of parallel alpha helices of varying lengths. There is only one section of Beta sheet longer than three residues in PAL, which resides in the funnel region leading to the active site. PAL and HAL (Histadine Ammonia Lyase) contain similar folds, but PAL differs from HAL with 215 additional residues. Of the 215 residues from this section, 155 residues extend above and below the main body of the structure, creating a "fan" arrangement, shown as the bracketed areas in Figure 3a.
 
 
 
 


==Central Core and MIO Cofactor==
==Central Core and MIO Cofactor==
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[[Image:Positive_Negative_Helices_PAL.png|thumb|left|Figure 6. Six positive poles toward the active site, one negative pole toward the active site]]
[[Image:Positive_Negative_Helices_PAL.png|thumb|left|Figure 6. Six positive poles toward the active site, one negative pole toward the active site]]




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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.
==Reaction Mechanism==
[[Image:L-phenylalanine.jpg|thumbnail|left|315px|Figure 10. Chemical Structure of L-Phenylalanine substrate]]
[[Image:Trans cinnamic acid.png|thumbnail|left|315px|Figure 12. ''trans''-cinnamic acid (CIN) is the product of the E1cB mechanism from L-Phe and PAL enzyme. CIN is an inhibitor to PAL. This is a useful negative feedback mechanism in living organisms so L-Phenylalanine reserves are not depleted via breakdown with PAL.]]
[[Image:E1cb_mechanism.png|thumbnail|right|600px|Figure 11. Elimination Unimolecular conjugate Base Mechanism showing carbanion intermediate. X is ammonia in this non-oxidative deamination reaction caused by the lyase.]]
[[Image:L-Phe_PAL_t-cinnamic_acid.png|thumbnail|right|600px|Figure 13. L-Phenylalanine substrate with k1 being the slow first step creating the stable carbanion, followed by a rapid product synsthesis in products CIN and Ammonia in Phenylalanine Ammonia Lyase enzyme.]]
<nowiki>'''Rate Law of E1cB Mechanism''': Second Order Kinetics observed</nowiki>
<nowiki>1) E1cB anion : anion is stable; rapid first step, followed by the slow formation of products (k1>>k2).</nowiki>
<nowiki>2) E1cB rev : first step is reversible, formation of product slower than reforming the starting material, this again results from a slow second step (k-1>>k2).</nowiki>
<nowiki>3) E1cB irr : first step is slow (formation of carbanion intermediate); but once formed, the product quickly follows (k2>>k1,k-1). This leads to an irreversible first step.</nowiki>


==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
'''H) Electron Density aps''': Using [X-Ray Diffraction] were displayed and models were manually constructed with the programs O (33) and XtalView/Xfit
'''I) Refinement Cycles''': performed with the program CNX, coordinates for the MIO moiety initially taken directly from the HAL structure
==PAL Biological Pathways and Implications==
PAL is found in mainly the lignin of higher plants, fungi, and yeast. In fungal and yeast cells, PAL is catabolic in generating carbon and nitrogen. In plants cells, PAL is used as the key biosynthetic enzyme is catalyzing the first synthesis step in polyphenyl compounds, as well as in defense mechanisms against ultra-violet light and herbivores.
Some examples of PAL in '''Metabolic Pathways''':
a) Tyrosine metabolism
b) Phenylalanine metabolism
c) Nitrogen metabolism
d) Phenylpropanoid biosynthesis
e) Alkaloid biosynthesis
PAL induces dramatically in response to various stimuli such as tissue wounding, pathogenic attack, light, low temperatures, and hormones.
'''PAL Substitution Therapy''':
PAL is now being used to treat patients with phenylketonuria (PKU). PKU is an autosomal recessive metabolic genetic disorder caused by mutation in phenylalanine hydroxylase (PAH) gene, which renders the enzyme that catalyzes amino acids phenylalanine and tyrosine nonfunctional. Conditions that come about from this disease are hyperphenylalaninemia (Elevation of Phenylalanine in the bloodstream) and mental retardation if therapy not begun at birth.
Instead of using natural formed PAL, doctors will substitute for a mutant recombinant PAL, which will decrease plasma levels of phenylalanine to harmless metabolites that can be excreted. The enzyme is modified to PEGylation to reduce immunogenicity; disguises introduced PAL from host’s immune system, leading to longer and more effective reduction in blood phenylalanine levels than non-modified PAL.<ref name="rutgers">http://maptest.rutgers.edu/drupal/?q=node/408</ref>
'''Aspartame Sweetener'''
A process developed by Genex Corporation in the United Kingdom using yeast Rhodotorula cells, the company utilizes the reverse reaction of the pathway, which requires two substrates. Conversion of trans-cinnamic acid to L-phenylalanine using reverse reaction, is catalyzed by PAL. L-Phenylalanine is a precursor for Aspartame, an artificial sweetener. <ref name="aspartame">http://www.aspartame.org/</ref>


==References==
==References==
<references />
<references />
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