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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.
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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[[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.]]
[[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>
 
 
 
 
 
 
 
 
 




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'''I) Refinement Cycles''': performed with the program CNX, coordinates for the MIO moiety initially taken directly from the HAL structure
'''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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