Tritium Secondary Kinetic Isotope Effect on Phenylalanine Ammonia-Lyase-Catalyzed Reaction

Lewandowicz, Andrzej; Jemielity, Jacek; Kańska, Marianna; Zoń, Jerzy; Paneth, Piotr

doi:10.1006/abbi.1999.1402

Cited by 17 publications

(14 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In the first 5 min, the k H /k T value was inverse (0.85), but after 20 min it became −normal× (1.15). The results of both groups [17] [18] support the involvement of the Ph group in the reaction mechanism, i.e., the FriedelÀCrafts-type attack at the Ph group (Scheme 3). It is to note that the D isotope effect shows the change in the overall reaction rate, while the T isotope effect, due to the trace labeling, is a measure for the isotope discrimination in the step in which T is involved.…”

Section: Results Andsupporting

confidence: 65%

See 1 more Smart Citation

Kinetic Analysis of the Reactions Catalyzed by Histidine and Phenylalanine Ammonia Lyases

Viergutz¹,

Rétey²

2004

Chemistry & Biodiversity

View full text Add to dashboard Cite

Although both the structures and the reactions of histidine and phenylalanine ammonia lyases (HAL and PAL) are very similar, the former shows a primary kinetic deuterium (D) isotope effect, while the latter does not. In the HAL reaction, the release of ammonia is partially rate-determining and is slower than the release of the product (E)-urocanate (4), whereas in the PAL reaction, the release of (E)-cinnamate (2) is the rate-limiting step. With (2S,3S)-[3-(2)H1]phenylalanine (1a), we determined the kinetic D isotope effects with the PAL mutants Q487A, Y350F, L137 H, and the double mutant L137 H/Q487E. The kH/kD values for the former two were of the same magnitude as with wild-type PAL (1.20+/-0.07), while the exchange of L137 to H almost doubled the effect (kH/kD=2.32+/-0.01). We conclude that L137 is part of the hydrophobic pocket harboring the phenyl group of the substrate/product and is responsible for its strong binding. The stability of the HAL ammonia complex was demonstrated 40 years ago. Here, we show that, in contrast to the former assumption, ammonia in the complex is not covalently bound to the prosthetic electrophile, 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO; 5). We carried out experiments with a mutant enzyme lacking MIO and exhibiting ca. 10(3) times less activity. Nevertheless, the enzyme-ammonia complex was formed, and the mutant behaved upon addition of (E)-[14C]urocanate (4a) like wild-type HAL. We conclude, therefore, that ammonia is bound in the complex by Coulomb forces as ammonium ion and can be released only after (E)-urocanate (4).

show abstract

Section: Results Andsupporting

confidence: 65%

“…Later, Paneth and co-workers measured the secondary tritium (T) isotope effect with [2, H]phenylalanine [18]. In the first 5 min, the k H /k T value was inverse (0.85), but after 20 min it became −normal× (1.15).…”

Section: Results Andmentioning

confidence: 97%

Kinetic Analysis of the Reactions Catalyzed by Histidine and Phenylalanine Ammonia Lyases

Viergutz¹,

Rétey²

2004

Chemistry & Biodiversity

View full text Add to dashboard Cite

show abstract

“…The Hanson and Havir's mechanism assumes that DHA plays both roles simultaneously, in contrary to the Shuster and Retey's mechanism, where DHA activates a hydrogen, while protonation of Phe is a separate step occurring upon. Also the data published of H/T KIE at ortho position of ring of l ‐Phe provides a strong argument for the Friedel–Crafts reaction type in the early steps of elimination of ammonia, which was confirmed by semi empiric calculation. For the confirmation of these hypotheses, the investigation of carbon KIE at the ortho position of l ‐Phe is needed.…”

Section: Resultsmentioning

confidence: 99%

Kinetic and solvent isotope effects on biotransformation of aromatic amino acids and their derivatives

Kańska

Jemielity

Pająk

et al. 2016

Labelled Comp Radiopharmac

Self Cite

View full text Add to dashboard Cite

Aromatic amino acids such as l-phenylalanine, l-tryptophan, 3',4'-dihydroxy-l-phenylalanine (l-DOPA), and their derivatives 3',4'-dihydroxyphenylacelaldehyde (DOPAL) and 3',4'-dihydroxyphenylethanol (DOPET), play an essential role in human metabolic processes. Incorrect or slow biotransformation of these compounds leads to some metabolic dysfunctions and in some cases to some neurodegenerative diseases. Therefore, studies of the biotransformation mechanisms of these metabolites draw biochemists' and medical researchers' attention. This study investigates the mechanisms of biotransformation of the aforementioned compounds using kinetic (KIE) and solvent (SIE) isotope effect methods. The overview presents the results and the numerical values of KIE and SIE methods, obtained in the study of biotransformation of l-phenylalanine, 5'-chloro-l-tryptophan, and l-DOPA, catalyzed by the enzymes from the lyases group (phenylalanine ammonia lyase, tryptophan indole-lyase, and tyrosine decarboxylase). Deuterium KIE was also determined during the deamination of 2'-chloro-l-phenylalanine in the presence of the enzyme l-phenylalanine dehydrogenase, as well as in the conversion of DOPAL into DOPET catalyzed by the enzyme alcohol dehydrogenase. The values of KIE and SIE have been determined using a noncompetitive spectrophotometric and a competitive (combined with internal radioactivity standard) radiometric methods.

show abstract

“…Further support for our mechanism was provided by recent publications. [20,21] Synthesis of and kinetic measurements with pyrimidinylalanines: The synthesis of b-(2-pyrimidinyl)-d,l-alanine (1) was carried out in six steps following the procedure of Haggerty et al [22] A chemoenzymatic strategy was applied to the preparation of b-(5-pyrimidinyl)-l-alanine (2). First, b-(5-pyrimidinyl)-acrylic acid (3) was synthesised starting from 5-bromo-pyrimidine and tert-butylacrylate under Heck conditions.…”

Section: Resultsmentioning

confidence: 99%

Phenylalanine Ammonia-Lyase: The Use of Its Broad Substrate Specificity for Mechanistic Investigations and Biocatalysis—Synthesis ofL-Arylalanines

et al. 2000

Self Cite

View full text Add to dashboard Cite

Several fluoro- and chlorophenylalanines were found to be good substrates of phenylalanine ammonia-lyase (PAL/EC 4.3.1.5) from parsley. The enantiomerically pure L-amino acids were obtained in good yields by reaction of the corresponding cinnamic acids with 5M ammonia solution (buffered to pH 10) in the presence of PAL. The kinetic constants for nine different fluoro- and chlorophenylalanines do not provide a rigorous proof for but are consistent with the previously proposed mechanism comprising an electrophilic attack of the methylidene-imidazolone cofactor of PAL at the aromatic nucleus as a first chemical step. In the resulting Friedel-Crafts-type sigma complex the beta-protons are activated for abstraction and consequently the pro-S is abstracted by an enzymic base. Results from semi-empirical calculations combined with a proposed partial active site model showed a correlation between the experimental kinetic constants and the change in polarization of the pro-S Cbeta-H bond and heat of formation of the sigma complexes, thus making the electrophilic attack at the neutral aromatic ring plausible. Furthermore, while 5-pyrimidinylalanine was found to be a moderately good substrate of PAL, 2-pyrimidinylalanine was an inhibitor.

show abstract

Tritium Secondary Kinetic Isotope Effect on Phenylalanine Ammonia-Lyase-Catalyzed Reaction

Cited by 17 publications

References 11 publications

Kinetic Analysis of the Reactions Catalyzed by Histidine and Phenylalanine Ammonia Lyases

Kinetic Analysis of the Reactions Catalyzed by Histidine and Phenylalanine Ammonia Lyases

Kinetic and solvent isotope effects on biotransformation of aromatic amino acids and their derivatives

Phenylalanine Ammonia-Lyase: The Use of Its Broad Substrate Specificity for Mechanistic Investigations and Biocatalysis—Synthesis ofL-Arylalanines

Contact Info

Product

Resources

About