Mechanism of action of urocanase

Klepp, Jürgen; Fallert‐Müller, Angelika; Grimm, K.; Hull, William E.; Rétey, János

doi:10.1111/j.1432-1033.1990.tb19274.x

Cited by 32 publications

(14 citation statements)

References 16 publications

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“…3). These considerations, combined with earlier results enlightening the mechanism of action of HAL and urocanase (33), where a transient electrophilic attack at the C-5 atom of imidazole modifies its reactivity, lead us to suggest an analogous role of the prosthetic dehydroalanine of PAL. This means an electrophilic attack at the phenyl group for which there is little if any precedence in enzymology.…”

Section: Discussionmentioning

confidence: 96%

The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

Schuster

Rétey

1995

Proc. Natl. Acad. Sci. U.S.A.

127

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Phenylalanine ammonia-lyase (EC 4.3.1.5) from parsley is posttranslationally modified by dehydrating its Ser-202 to the catalytically essential dehydroalanine prosthetic group. The codon of Ser-202 was changed to those of alanine and threonine by site-directed mutagenesis. These mutants and the recombinant wild-type enzyme, after treatment with sodium borohydride, were virtually inactive with L-phenylalanine as substrate but catalyzed the deamination of L-4-nitrophenylalanine, which is also a substrate for the wild-type enzyme. Although the mutants reacted about 20 times slower with L-4-nitrophenylalanine than the wild-type enzyme, their V... for L-4-nitrophenylalanine was two orders of magnitude higher than for L-phenylalanine. In contrast to L-tyrosine, which was a poor substrate, DL-3-hydroxyphenylalanine (DL-m-tyrosine) was converted by phenylalanine ammonia-lyase at a rate comparable to that of L-phenylalanine. These results suggest a mechanism in which the crucial step is an electrophilic attack of the prosthetic group at position 2 or 6 of the phenyl group. In the resulting carbenium ion, the 18-Hsi atom is activated in a similar way as it is in the nitro analogue. Subsequent elimination of ammonia, concomitant with restoration of both the aromatic ring and the prosthetic group, completes the catalytic cycle.Phenylalanine ammonia-lyase (PAL) is an important plant enzyme that converts L-phenylalanine into trans-cinnamic acid, which in turn is the precursor of various phenylpropanoids, such as lignins, flavonoids, and coumarins (1, 2). It has been known for a long time that PAL contains a catalytically essential dehydroalanine residue (3). Although the latter occurs in a number of natural peptides (4, 5) and has been postulated as an intermediate in the conversion of L-serine residues into D-alanine (6), it is shared only by one related enzyme, histidine ammonia-lyase (HAL) (7-10).The electrophilic property of the prosthetic dehydroalanine has been demonstrated by addition of various nucleophiles, such as nitromethane (7-11), cyanide (12), and sodium borohydride (3,8). All these reagents caused inactivation and, using radiolabel, incorporation of the radioisotope into the expected product. Twenty-five years ago a mechanism was proposed for the PAL and HAL reactions involving as an initial step the nucleophilic attack at the dehydroalanine residue by the amino group of the substrate (3, 7). Such a reaction should enhance the leaving ability of the amino group. Recently, this laboratory has identified the precursor of the dehydroalanine in HAL from Pseudomonas putida and PAL from parsley to be Ser-143 and Ser-202, respectively (9, 10, 13). Heterologous expression allowed the production of mutants by site-directed mutagenesis. The PAL mutant S202A and the HAL mutants S143A and S143T were virtually inactive with L-phenylalanine and L-histidine as substrates, respectively (9,10,13,14). Surprisingly, L-5-nitrohistidine, known to be a moderately good substrate of HAL, reacted with the dehydroalanine-less m...

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Section: Discussionmentioning

confidence: 96%

The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

Schuster

Rétey

1995

Proc. Natl. Acad. Sci. U.S.A.

127

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“…The imidazole unit is biodegradable through oxidation (enzymatic or ROS), leading to scission of the imidazole ring. This ring scission produces amino acids and amides, which can be metabolized by the respective organism . It is difficult to find representatives of conjugated polymers in the realms of natural materials; however, melanin can be considered a conjugated polymer despite its ill‐defined and crosslinked nature.…”

Section: Clearance Of Conjugated Polymer Particlesmentioning

confidence: 99%

Conjugated Polymer Nanoparticles toward In Vivo Theranostics – Focus on Targeting, Imaging, Therapy, and the Importance of Clearance

Kuehne

2017

Adv. Biosys.

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Conjugated polymer nanoparticles are highly fluorescent colloids with tunable emission colors ranging from the visible deep into the near infrared spectrum. Conjugated polymer nanoparticles are easy to prepare, tunable in their size, and virtually nonbleachable. Conjugated polymer particles can also be designed to give off heat upon irradiation. All these properties make conjugated polymer particles ideal materials for biomedical fluorescence and photoacoustic imaging as well as for theranostic applications. Here, different examples of surface functionalization to attach pathological homing devices, imaging modalities, as well as the emerging possibilities for therapeutic measures are discussed. Furthermore, clearance of the particles is considered, which is important to ultimately apply the materials for in vivo theranostics. Due to the conjugated backbone of the conjugated polymers, established degradation strategies, as known from hydrophilic nonconjugated polymer carriers, cannot be applied. Bioinspired strategies and potential pathways for degradation and clearance via structural changes upon triggers such as pH, oxidation, and temperature are also discussed in this progress report.

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“…Since soaking experiments with the inhibitor imidazolyl-propionate, which is urocanate with a reduced C a -C b double bond, 22 yielded no additional density at the active center of the urocanase-S crystals, we tried to soak an inactivated enzyme with the substrate urocanate. For this purpose we removed the arginine side-chain contacting the nicotinamide by the mutation Arg455/ Ala, which destroyed the enzyme activity.…”

Section: Structure Analysismentioning

confidence: 99%