PAL

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Phenylalanine ammonia-lyase (PAL) is an enzyme that catalyzes the direct removal of ammonia on L-phenylalanine to produce trans-cinnamic acid. Found in barley. Exist in the soluble part of higher plants, yeasts, and fungi. The estimated molecular weight is 300,000. This is an enzyme that can use phenylalanine for the synthesis of phenolic compounds. In many cases, the reaction becomes a stepwise rate stage in the synthesis of phenolic compounds. The activity in tissues can change significantly with external factors, and treatment with light, disease, and plant hormones will increase the activity significantly. In addition, it is sometimes dominated by photosensitive pigments. In most cases, the enzyme inactivation occurs while the activity in the tissue increases. At this time, the amount of active enzyme in the tissue will soon decrease. It is believed that this inactivation is related to the action of protein-like substances. In addition, the presence of inactive enzymes has also been pointed out.

Figure 1. Protein structure of PAL.

Structure

Phenylalanine ammonia lyase consists of four identical subunits, mainly composed of α-helices, and pairs of monomers form a single active site. The catalytic effect in PAL may be determined by the dipole moments of seven different alpha helices associated with the active site. 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 helix, while Phe413, Glu496 and Gln500 contribute to the stability of MIO cofactors. The direction of the dipole moment generated by the helix in the active site creates a positively charged region to ideally react with MIO. Part of the positive area of the active site may also help stabilize the charge of the carbanion intermediate. PAL is structurally similar to the mechanically related histidine ammonia lyase, although PAL also has approximately 215 residues.

Functions

Phenylalanine ammonia lyase can perform different functions in different species. Mainly found in certain plants and fungi (ie yeast). In fungi and yeast cells, PAL plays an important catabolism effect, producing carbon and nitrogen. In plants, PAL is an important biosynthetic enzyme, which catalyzes the first step in the synthesis of a variety of polyphenylene compounds and mainly participates in defense mechanisms. PAL is involved in 5 metabolic pathways: tyrosine metabolism, phenylalanine metabolism, nitrogen metabolism, phenylpropane biosynthesis and alkaloid biosynthesis.

Mechanism

Phenylalanine ammonia lyase is specific for L-phe and to a lesser extent specific for L-tyrosine. The reaction catalyzed by PAL is a spontaneous elimination reaction, not an oxidative deamination reaction.

L-phenylalanine→trans-cinnamic acid + NH₃

The cofactor 3,5-dihydro-5-methyldiene-4H-imidazol-4-one (MIO) participates in the reaction and is located above the positive pole of the three polar helices in the active site, which helps increase its Electrophilic. MIO is attacked by the aromatic ring of L-phe, which activates the C-H bond on the β carbon, deprotonating basic residues. The E1cB eliminates the carbanion intermediate of the reaction (stabilized by the partial positive region of the active site), and then discharges ammonia to form cinnamic acid olefins. It is believed that the reaction mechanism of PAL is similar to that of the related enzyme histidine ammonia lyase. For a long time, dehydroalanine residues were considered to be the key electrophilic catalytic residues in PAL and HAL, but the active residues were later discovered. It's MIO, more electrophilic. It is formed by the cyclization and dehydration of the conservative Ala-Ser-Gly tripeptide fragment.