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:<small>L</small>-phenylalanine <math>\rightleftharpoons</math> ''trans''-cinnamate + NH<sub>3</sub>
:<small>L</small>-phenylalanine <math>\rightleftharpoons</math> ''trans''-cinnamate + NH<sub>3</sub>


The [[cofactor (biochemistry)|cofactor]] [[3,5-dihydro-5-methyldiene-4H-imidazol-4-one|3,5-dihydro-5-methyldiene-4''H''-imidazol-4-one]] (MIO) is involved in the reaction and sits atop the positive pole of three polar helices in the active site, which helps to increase its [[electrophile|electrophilicity]].<ref name="Alunni">{{cite journal|last=Alunni|first=S|author2=Cipiciani, A |author3=Fioroni, G |author4= Ottavi, L |title=Mechanisms of inhibition of phenylalanine ammonia-lyase by phenol inhibitors and phenol/glycine synergistic inhibitors|journal=Archives of Biochemistry and Biophysics|date=Apr 15, 2003|volume=412|issue=2|pages=170–5|pmid=12667480|doi=10.1016/s0003-9861(03)00007-9}}</ref> MIO is attacked by the [[aromatic hydrocarbon|aromatic ring]] of <small>L</small>-phe, which activates the C-H bond on the β carbon for deprotonation by a [[base (chemistry)|basic]] residue.<ref name="Langer">{{cite journal|last=Langer|first=B|author2=Langer, M |author3=Rétey, J |title=Methylidene-imidazolone (MIO) from histidine and phenylalanine ammonia-lyase.|journal=Advances in protein chemistry|year=2001|volume=58|pages=175–214|pmid=11665488|doi=10.1016/s0065-3233(01)58005-5}}</ref><ref>{{cite book |title= Enzymatic Reaction Mechanisms |first1= Perry A. |last1= Frey |first2= Adrian D. |last2= Hegeman |chaptertitle= Methylene imidazolone-dependent elimination and addition: phenylalanine ammonia-lyase |pages= 460–466 |publisher= Oxford University Press |year= 2007 |isbn= 9780195352740 }}</ref> The [[carbanion]] [[reaction intermediate|intermediate]] of this [[E1cB-elimination reaction]], which is stabilized by partial positive regions in the active site, then expels ammonia to form the cinnamate alkene. The mechanism of the reaction of PAL is thought to be similar to the mechanism of the related enzyme histidine ammonia lyase.<ref name="Langer"/> PAL is inhibited by trans-cinnamic acid, and, in some species, may be inhibited by ''trans-''cinnamic acid derivatives.<ref name="first"/><ref>{{cite journal|last=Sato|first=Toshitsugu|author2=Kiuchi, Fumiyuki |author3=Sankawa, Ushio |title=Inhibition of phenylalanine ammonia-lyase by cinnamic acid derivatives and related compounds|journal=Phytochemistry|date=1 January 1982|volume=21|issue=4|pages=845–850|doi=10.1016/0031-9422(82)80077-0}}</ref> The [[unnatural amino acid]]s <small>D</small>-Phe and <small>D</small>-Tyr, the [[enantiomer]]ic forms of the normal substrate, are [[competitive inhibition|competitive inhibitors]].<ref name="Hodgins2"/>
The [[cofactor (biochemistry)|cofactor]] [[3,5-dihydro-5-methyldiene-4H-imidazol-4-one|3,5-dihydro-5-methyldiene-4''H''-imidazol-4-one]] (MIO) is involved in the reaction and sits atop the positive pole of three polar helices in the active site, which helps to increase its [[electrophile|electrophilicity]].<ref name="Alunni">{{cite journal|last=Alunni|first=S|author2=Cipiciani, A |author3=Fioroni, G |author4= Ottavi, L |title=Mechanisms of inhibition of phenylalanine ammonia-lyase by phenol inhibitors and phenol/glycine synergistic inhibitors|journal=Archives of Biochemistry and Biophysics|date=Apr 15, 2003|volume=412|issue=2|pages=170–5|pmid=12667480|doi=10.1016/s0003-9861(03)00007-9}}</ref> MIO is attacked by the [[aromatic hydrocarbon|aromatic ring]] of <small>L</small>-phe, which activates the C-H bond on the β carbon for deprotonation by a [[base (chemistry)|basic]] residue.<ref name="Langer">{{cite journal|last=Langer|first=B|author2=Langer, M |author3=Rétey, J |title=Methylidene-imidazolone (MIO) from histidine and phenylalanine ammonia-lyase.|journal=Advances in protein chemistry|year=2001|volume=58|pages=175–214|pmid=11665488|doi=10.1016/s0065-3233(01)58005-5}}</ref><ref>{{cite book |title= Enzymatic Reaction Mechanisms |first1= Perry A. |last1= Frey |first2= Adrian D. |last2= Hegeman |chapter= Methylene imidazolone-dependent elimination and addition: phenylalanine ammonia-lyase |pages= 460–466 |publisher= Oxford University Press |year= 2007 |isbn= 9780195352740 }}</ref> The [[carbanion]] [[reaction intermediate|intermediate]] of this [[E1cB-elimination reaction]], which is stabilized by partial positive regions in the active site, then expels ammonia to form the cinnamate alkene. The mechanism of the reaction of PAL is thought to be similar to the mechanism of the related enzyme histidine ammonia lyase.<ref name="Langer"/> PAL is inhibited by trans-cinnamic acid, and, in some species, may be inhibited by ''trans-''cinnamic acid derivatives.<ref name="first"/><ref>{{cite journal|last=Sato|first=Toshitsugu|author2=Kiuchi, Fumiyuki |author3=Sankawa, Ushio |title=Inhibition of phenylalanine ammonia-lyase by cinnamic acid derivatives and related compounds|journal=Phytochemistry|date=1 January 1982|volume=21|issue=4|pages=845–850|doi=10.1016/0031-9422(82)80077-0}}</ref> The [[unnatural amino acid]]s <small>D</small>-Phe and <small>D</small>-Tyr, the [[enantiomer]]ic forms of the normal substrate, are [[competitive inhibition|competitive inhibitors]].<ref name="Hodgins2"/>


==Enzyme structure==
==Enzyme structure==

Revision as of 21:14, 3 May 2018

phenylalanine ammonia-lyase
PDB rendering based on 1T6J.
Identifiers
EC no.4.3.1.24
CAS no.9024-28-6
Databases
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KEGGKEGG entry
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Phenylalanine ammonia lyase (EC 4.3.1.24) is an enzyme that catalyzes a reaction converting L-phenylalanine to ammonia and trans-cinnamic acid.[1] Phenylalanine ammonia lyase (PAL) is the first and committed step in the phenyl propanoid pathway and is therefore involved in the biosynthesis of the polyphenol compounds such as flavonoids, phenylpropanoids, and lignin in plants.[2][3] Phenylalanine ammonia lyase is found widely in plants, as well as some bacteria, yeast and fungi, with isoenzymes existing within many different species. It has a molecular mass in the range of 270–330 kDa.[1][4] The activity of PAL is induced dramatically in response to various stimuli such as tissue wounding, pathogenic attack, light, low temperatures, and hormones.[1][5] PAL has recently been studied for possible therapeutic benefits in humans afflicted with phenylketonuria.[6] It has also been used in the generation of L-phenylalanine as precursor of the sweetener aspartame.[7]

The enzyme is a member of the ammonia lyase family, which cleaves carbon-nitrogen bonds. Like other lyases, phenylalanine requires only one substrate for the forward reaction, but two for the reverse. It is thought to be mechanistically similar to the related enzyme histidine ammonia-lyase (EC:4.3.1.3, HAL).[8] The systematic name of this enzyme class is L-phenylalanine ammonia-lyase (trans-cinnamate-forming). Previously, it was designated EC 4.3.1.5, but that class has been redesignated as EC 4.3.1.24 (phenylalanine ammonia-lyases), EC 4.3.1.25 (tyrosine ammonia-lyases), and EC 4.3.1.26 (phenylalanine/tyrosine ammonia-lyases). Other names in common use include tyrase, phenylalanine deaminase, tyrosine ammonia-lyase, L-tyrosine ammonia-lyase, phenylalanine ammonium-lyase, PAL, and L-phenylalanine ammonia-lyase.

Enzyme mechanism

Phenylalanine ammonia lyase is specific for L-phe, and to a lesser extent, L-tyrosine.[9][10] The reaction catalyzed by PAL is the spontaneous, non-oxidative deamination of L-phenylalanine to yield trans-cinnamic acid and ammonia.[11]

L-phenylalanine trans-cinnamate + NH3

The cofactor 3,5-dihydro-5-methyldiene-4H-imidazol-4-one (MIO) is involved in the reaction and sits atop the positive pole of three polar helices in the active site, which helps to increase its electrophilicity.[12] MIO is attacked by the aromatic ring of L-phe, which activates the C-H bond on the β carbon for deprotonation by a basic residue.[13][14] The carbanion intermediate of this E1cB-elimination reaction, which is stabilized by partial positive regions in the active site, then expels ammonia to form the cinnamate alkene. The mechanism of the reaction of PAL is thought to be similar to the mechanism of the related enzyme histidine ammonia lyase.[13] PAL is inhibited by trans-cinnamic acid, and, in some species, may be inhibited by trans-cinnamic acid derivatives.[1][15] The unnatural amino acids D-Phe and D-Tyr, the enantiomeric forms of the normal substrate, are competitive inhibitors.[9]

Enzyme structure

PAL active site

Phenylalanine ammonia lyase is composed of four identical subunits composed mainly of alpha-helices, with pairs of monomers forming a single active site.[16] Catalysis in PAL may be governed by the dipole moments of seven different alpha helices associated with the active site.[17] The active site contains the electrophilic group MIO non-covalently bonded to three helices. Leu266, Asn270, Val269, Leu215, Lys486, and Ile472 are located on the active site helices, while Phe413, Glu496, and Gln500 contribute to the stabilization of the MIO cofactor. The orientation of dipole moments generated by helices within the active site generates an electropositive region for ideal reactivity with MIO. The partially positive regions in the active site may also help stabilize the charge of a carbanion intermediate. PAL is structurally similar to the mechanistically related histidine ammonia lyase, although PAL has approximately 215 additional residues.[16]

Biological function

Phenylalanine ammonia lyase can perform different functions in different species. It is found mainly in some plants and fungi (i.e. yeast). In fungal and yeast cells, PAL plays an important catabolic role, generating carbon and nitrogen.[2] In plants it is a key biosynthetic enzyme that catalyzes the first step in the synthesis of a variety of polyphenyl compounds [2][3] and is mainly involved in defense mechanisms. PAL is involved in 5 metabolic pathways: tyrosine metabolism, phenylalanine metabolism, nitrogen metabolism, phenylpropanoid biosynthesis, and alkaloid biosynthesis ii.

Disease relevance

Researchers are exploring enzyme substitution therapy using PAL to treat phenylketonuria (PKU), an autosomal recessive genetic disorder in humans in which mutations in the phenylalanine hydroxylase (PAH, EC 1.14.16.1) gene inactivate the enzyme.[6] This leads to an inability of the patient to metabolize phenylalanine, causing elevated levels of Phe in the bloodstream (hyperphenylalaninemia) and mental retardation if therapy is not begun at birth.[6] Treatment in mouse models using recombinant PAL has been shown to lower plasma Phe level, converting L-phe to harmless metabolites that can be excreted. In order to reduce the immunogenicity of PAL, the enzyme is modified by PEGylation. This process disguises the introduced PAL from the host’s immune system and can lead to a longer and more effective reduction in blood Phe levels than non-modified PAL.[18]

Current treatment for PKU requires patients to adhere to a specific diet their whole life, which can be difficult to maintain and may contribute to birth defects if pregnant patients are not careful enough.[19] Therefore, continued research into the development of enzyme therapy using PAL is of interest for the medical community.

PAL may also be used in the treatment of certain forms of mouse neoplastic tumors.[2]

Industrial relevance

Artificial sweeteners

The reverse reaction catalyzed by PAL has been used to convert trans-cinnamic acid to L-phenylalanine, which is a precursor of the sweetener aspartame. This process was developed by Genex Corporation in the USA using yeast Rhodotorula cells (U.S. patent 4,574,117; U.S. patent 4,584,269; U.S. patent 4,584,273; U.S. patent 4,598,047; U.S. patent 4,636,466; U.S. patent 4,681,850; U.S. patent 4,757,015).[7]

Unnatural amino acid synthesis

Analogous to how aspartame is synthesized, PAL is also used to synthesize unnatural amino acids from various substituted cinnamic acids for research purposes.[20] Steric hindrance from arene substitution limits PAL's utility for this purpose however.[21] For instance, when Rhodotorula glutinis was used to affect this biotransformation the enzyme was discovered to be intolerant of all para substituents other than F, presumably due to the element's small atomic radius. Meta and ortho positions were found to be more tolerant, but still limited by, larger substituents. For instance the enzyme's active site permitted ortho methoxy substitution but forbade meta ethoxy. Other organisms with different versions of the enzyme may be less limited in this way.[22][23]

Structural studies

As of late 2007, 5 structures have been solved for this class of enzymes, with PDB accession codes 1T6J, 1T6P, 1W27, 1Y2M, and 2NYF.

Notes

  1. ^ a b c d Camm, Edith L.; Towers, G.H.Neil (1 May 1973). "Phenylalanine ammonia lyase". Phytochemistry. 12 (5): 961–973. doi:10.1016/0031-9422(73)85001-0.
  2. ^ a b c d Fritz, RR; Hodgins, DS; Abell, CW (Aug 10, 1976). "Phenylalanine ammonia-lyase. Induction and purification from yeast and clearance in mammals". The Journal of Biological Chemistry. 251 (15): 4646–50. PMID 985816.
  3. ^ a b Tanaka, Y; Matsuoka, M; Yamanoto, N; Ohashi, Y; Kano-Murakami, Y; Ozeki, Y (August 1989). "Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato". Plant Physiology. 90 (4): 1403–7. doi:10.1104/pp.90.4.1403. PMC 1061903. PMID 16666943.
  4. ^ Appert, C; Logemann, E; Hahlbrock, K; Schmid, J; Amrhein, N (Oct 1, 1994). "Structural and catalytic properties of the four phenylalanine ammonia-lyase isoenzymes from parsley (Petroselinum crispum Nym.)". European Journal of Biochemistry / FEBS. 225 (1): 491–9. doi:10.1111/j.1432-1033.1994.00491.x. PMID 7925471.
  5. ^ Hahlbrock, K; Grisebach, H (1 June 1979). "Enzymic Controls in the Biosynthesis of Lignin and Flavonoids". Annual Review of Plant Physiology. 30 (1): 105–130. doi:10.1146/annurev.pp.30.060179.000541.
  6. ^ a b c Sarkissian, Christineh N.; Gámez, Alejandra (1 December 2005). "Phenylalanine ammonia lyase, enzyme substitution therapy for phenylketonuria, where are we now?". Molecular Genetics and Metabolism. 86: 22–26. doi:10.1016/j.ymgme.2005.06.016. PMID 16165390.
  7. ^ a b Evans, ChristopherT.; Hanna, Kim; Conrad, Dayle; Peterson, Wendy; Misawa, Masanaru (1 February 1987). "Production of phenylalanine ammonia-lyase (PAL): isolation and evaluation of yeast strains suitable for commercial production of L-phenylalanine". Applied Microbiology and Biotechnology. 25 (5). doi:10.1007/BF00253309.
  8. ^ Schwede, TF; Rétey, J; Schulz, GE (Apr 27, 1999). "Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile". Biochemistry. 38 (17): 5355–61. doi:10.1021/bi982929q. PMID 10220322.
  9. ^ a b Hodgins, DS (May 10, 1971). "Yeast phenylalanine ammonia-lyase. Purification, properties, and the identification of catalytically essential dehydroalanine". The Journal of Biological Chemistry. 246 (9): 2977–85. PMID 5102931.
  10. ^ Barros, J.; Serrani-Yarce, J.C.; Chen, F.; Baxter, D.; Venables, B.J.; Dixon, R.A. (2016). "Role of bifunctional ammonia-lyase in grass cell wall biosynthesis". Nat. Plants. 2: 16050. doi:10.1038/nplants.2016.50. PMID 27255834.
  11. ^ Koukol, J; Conn, EE (October 1961). "The metabolism of aromatic compounds in higher plants. IV. Purification and properties of the phenylalanine deaminase of Hordeum vulgare". The Journal of Biological Chemistry. 236: 2692–8. PMID 14458851.
  12. ^ Alunni, S; Cipiciani, A; Fioroni, G; Ottavi, L (Apr 15, 2003). "Mechanisms of inhibition of phenylalanine ammonia-lyase by phenol inhibitors and phenol/glycine synergistic inhibitors". Archives of Biochemistry and Biophysics. 412 (2): 170–5. doi:10.1016/s0003-9861(03)00007-9. PMID 12667480.
  13. ^ a b Langer, B; Langer, M; Rétey, J (2001). "Methylidene-imidazolone (MIO) from histidine and phenylalanine ammonia-lyase". Advances in protein chemistry. 58: 175–214. doi:10.1016/s0065-3233(01)58005-5. PMID 11665488.
  14. ^ Frey, Perry A.; Hegeman, Adrian D. (2007). "Methylene imidazolone-dependent elimination and addition: phenylalanine ammonia-lyase". Enzymatic Reaction Mechanisms. Oxford University Press. pp. 460–466. ISBN 9780195352740.
  15. ^ Sato, Toshitsugu; Kiuchi, Fumiyuki; Sankawa, Ushio (1 January 1982). "Inhibition of phenylalanine ammonia-lyase by cinnamic acid derivatives and related compounds". Phytochemistry. 21 (4): 845–850. doi:10.1016/0031-9422(82)80077-0.
  16. ^ a b Calabrese, JC; Jordan, DB; Boodhoo, A; Sariaslani, S; Vannelli, T (Sep 14, 2004). "Crystal structure of phenylalanine ammonia lyase: multiple helix dipoles implicated in catalysis". Biochemistry. 43 (36): 11403–16. doi:10.1021/bi049053+. PMID 15350127.
  17. ^ Pilbák, S; Tomin, A; Rétey, J; Poppe, L (March 2006). "The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases". The FEBS Journal. 273 (5): 1004–19. doi:10.1111/j.1742-4658.2006.05127.x. PMID 16478474.
  18. ^ Gámez, A; Wang, L; Straub, M; Patch, MG; Stevens, RC (January 2004). "Toward PKU enzyme replacement therapy: PEGylation with activity retention for three forms of recombinant phenylalanine hydroxylase". Molecular Therapy. 9 (1): 124–9. doi:10.1016/j.ymthe.2003.11.002. PMID 14741785.
  19. ^ Lenke, RR; Levy, HL (Mar 1, 1982). "Maternal phenylketonuria--results of dietary therapy". American Journal of Obstetrics and Gynecology. 142 (5): 548–53. doi:10.1016/0002-9378(82)90759-1. PMID 7058857.
  20. ^ Hughes, Andrew (2009). Amino Acids, Peptides and Proteins in Organic Chemistry Volume 1. Weinheim Germany: Wiley VCH. p. 94. ISBN 9783527320967.
  21. ^ Renard, G; Guilleux, J; Bore, C; Malta-Valette, V; Lerner, D (1992). "Synthesis of L-phenylalanine analogs by Rhodotorula glutinis. Bioconversion of cinnamic acids derivatives". Biotechnology Letters. 14 (8): 673–678. doi:10.1007/BF01021641.
  22. ^ Lovelock, Sarah L.; Turner, Nicholas J. (2014). "Bacterial Anabaena variabilis phenylalanine ammonia lyase: A biocatalyst with broad substrate specificity". Bioorganic & Medicinal Chemistry. 22 (20): 5555–5557. doi:10.1016/j.bmc.2014.06.035. PMID 25037641.
  23. ^ Showa, Denko; Hirobumi, Aoki. "Production process of L-phenylalanine derivatives by microorganisms". Google Patents. Hirobumi Central Research Laboratory. Retrieved 20 July 2014.

References

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