Inhibition of histidine ammonia lyase by 8-methoxypsoralen and psoralen-oxidized photoproducts.

The effect of 8-methoxypsoralen-UVA therapy on the catalysis of histidine to trans-urocanic acid by histidine ammonia lyase (HAL, EC 4.3.1.3) was examined using an enzymatic assay from Sigma-Aldrich where the growth of the trans-urocanic acid peak at 277 nm was monitored. A Rayonet Photochemical Mini Reactor (Model RMR-600) equipped with eight, 3500 Å light sources and a custom UVA filter (Model S-BAL3 2.9 mm), from the Solar Light Company, were used to expose various reaction mixtures to broadband UVA light and UVA/UVB light. A UV-Vis spectrophotometer (Model Shimadzu UV 2540) with a temperature-controlled cell holder (Model TCC240) was used to monitor the growth of the trans-urocanic peak. Results of dark-binding experiments of 8-methoxypsoralen in denatured ethanol indicate no inhibition of enzyme activity due to ethanol but noncompetitive inhibition due to 8-methoxypsoralen. The effects of preirradiated 8-methoxypsoralen, with both broadband UVA and UVA/UVB, indicate that inhibition was due to psoralen-oxidized photoproducts. Inhibition of HAL was found when exposed to broadband UVA/UVB and to a lesser extent when exposed to broadband UVA.

Inhibition of histidine ammonia lyase by heteroaryl-alanines and acrylates.

Histidine ammonia lyase (HAL) catalyzes the elimination of ammonia from the substrate to form (E)-urocanate. The interaction between HAL and acrylic acids or alanines substituted with heteroaryl groups in the beta-position was investigated. These proved to be strong competitive inhibitors when the heteroaryl groups were furanyl, thiophenyl, benzofuranyl, and benzothiophenyl, carrying the alanyl or acrylic side chains either in 2 or 3 positions, with K(i) values between 18 and 139 microM. The exception was (furan-3-yl)alanine which was found to be inert. Tryptophan and 1-methyltryptophan, as well as the corresponding acrylates (=prop-2-enoates), are strong mixed inhibitors of HAL. Theoretically, L-histidine can be dissected into 4-methyl-1H-imidazole and glycine. Whereas these two compounds separately are only very weak inhibitors of HAL, equimolar amounts of both show a K(i) value of 1.7+/-0.09 mM which is to be compared with the K(m) value of 15.6 mM for the normal reaction. We conclude that 5-methyl-1H-imidazole and glycine mimic the substrate and occupy the active site of HAL in a similar orientation.

Structures of two histidine ammonia-lyase modifications and implications for the catalytic mechanism.

Histidine ammonia-lyase (EC 4.3.1.3) catalyzes the nonoxidative elimination of the alpha-amino group of histidine using a 4-methylidene-imidazole-5-one (MIO), which is formed autocatalytically from the internal peptide segment 142Ala-Ser-Gly. The structure of the enzyme inhibited by a reaction with l-cysteine was established at the very high resolution of 1.0 A. Five active center mutants were produced and their catalytic activities were measured. Among them, mutant Tyr280-->Phe could be crystallized and its structure could be determined at 1.7 A resolution. It contains a planar sp2-hybridized 144-N atom of MIO, in contrast to the pyramidal sp3-hybridized 144-N of the wild-type. With the planar 144-N atom, MIO assumes the conformation of a putative intermediate aromatic state of the reaction, demonstrating that the conformational barrier between aromatic and wild-type states is very low. The data led to a new proposal for the geometry for the catalyzed reaction, which also applies to the closely related phenylalanine ammonia-lyase (EC 4.3.1.5). Moreover, it suggested an intermediate binding site for the released ammonia.

Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida.

Elucidation of the 3D structure of histidine ammonia-lyase (HAL, EC 4.3.1.3) from Pseudomonas putida by X-ray crystallography revealed that the electrophilic prosthetic group at the active site is 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) [Schwede, T.F., Rétey, J., Schulz, G.E. (1999) Biochemistry, 38, 5355-5361]. To evaluate the importance of several amino-acid residues at the active site for substrate binding and catalysis, we mutated the following amino-acid codons in the HAL gene: R283, Y53, Y280, E414, Q277, F329, N195 and H83. Kinetic measurements with the overexpressed mutants showed that all mutations resulted in a decrease of catalytic activity. The mutants R283I, R283K and N195A were approximately 1640, 20 and 1000 times less active, respectively, compared to the single mutant C273A, into which all mutations were introduced. Mutants Y280F, F329A and Q277A exhibited approximately 55, 100 and 125 times lower activity, respectively. The greatest loss of activity shown was in the HAL mutants Y53F, E414Q, H83L and E414A, the last being more than 20 900-fold less active than the single mutant C273A, while H83L was 18 000-fold less active than mutant C273A. We propose that the carboxylate group of E414 plays an important role as a base in catalysis. To investigate a possible participation of active site amino acids in the formation of MIO, we used the chromophore formation upon treatment of HAL with l-cysteine and dioxygen at pH 10.5 as an indicator. All mutants, except F329A showed the formation of a 338-nm chromophore arising from a modified MIO group. The UV difference spectra of HAL mutant F329A with the MIO-free mutant S143A provide evidence for the presence of a MIO group in HAL mutant F329A also. For modelling of the substrate arrangement within the active site and protonation state of MIO, theoretical calculations were performed.

On the nature of the irreversible inhibition of histidine ammonia lyase by cysteine and dioxygen.

The irreversible inhibition of histidine ammonia lyase by L-cysteine and dioxygen has been reexamined. After denaturation and consecutive digestion of the inhibited enzyme by trypsin and endoproteinase Glu-C, the generated chromophoric system (lambda max = 340 nm) remained intact and was isolated in an octapeptide containing amino acids 138-145, as previously described (Hernandez, D.; Stroh, J.G.; Phillips, A. T. Arch. Biochem. Biophys. 1993, 307, 126). Conducting the inhibition in the presence of 18O2 did not result in the incorporation of the heavy isotope into the isolated octapeptide. Total hydrolysis followed by amino acid analysis of the octapeptide revealed the presence of one arginine in addition to those expected from the deduced sequence (G3SVAD). 1H NMR spectroscopy of the octapeptide confirmed the presence of the amino acids GSVAD and showed no signals for olefinic or aromatic protons. To account for the excess mass of the octapeptide, we propose an oxidative degradation of the dehydroalanine prosthetic group, followed by reaction of the resulting dicarbonyl system with a nearby arginine residue.

Histidine ammonia-lyase mutant S143C is posttranslationally converted into fully active wild-type enzyme. Evidence for serine 143 to be the precursor of active site dehydroalanine.

Histidase [histidine ammonia-lyase (HAL); EC 4.3.1.3] from Pseudomonas putida is a homotetramer and contains one catalytically essential dehydroalanine residue per subunit. Since the mutant S143A was catalytically inert, it has been proposed that serine 143 is the precursor of the active site dehydroalanine [Langer et al. (1994) Biochemistry 33, 6462-6467]. To further define the role of serine 143, we prepared the mutants S143T and S143C by site-directed mutagenesis. The threonine 143 mutant was neither catalytically active (< 0.01%) nor did it form with L-cysteine and oxygen a product absorbing at 340 nm. In contrast, the cysteine 143 mutant showed full catalytic activity and, after treatment with L-cysteine and oxygen, an increased absorbance at 340 nm similar to that of the wild-type enzyme. Also the kinetic constants (Km and Vmax) were identical with those of wild-type histidase. Titration with Ellman's reagent revealed that both wild-type and S143C mutant histidase contained seven thiol groups after exhaustive reduction. It must be concluded that posttranslational modification occurs with both serine 143 and cysteine 143 by elimination of water and hydrogen sulfide, respectively. In both cases dehydroalanine is formed and the resulting histidases are indistinguishable. In contrast, the threonine 143 mutant is not processed to active enzyme.

1-amino-2-imidazol-4'-ylethylphosphonic acid is a potent reversible inhibitor of Pseudomonas putida histidine ammonia-lyase.

A phosphonic acid analogue of L-histidine, 1-amino-2-imidazol-4'-ylethylphosphonic acid (HisP), was identified as a reversible competitive inhibitor of histidine ammonia-lyase (histidase). The affinity of histidase for HisP was pH dependent, with Ki values of 0.28 microM and 10.4 microM compared to substrate Km values of 1 and 5 mM at pH 7 and 9, respectively. HisP did not appear to be a substrate for histidase. A twenty-fold molar excess of HisP over enzyme completely protected the active site of histidase from inactivation by 10 mM bisulfite. Neither isohistidine nor two other phosphonic acid compounds were inhibitory towards histidase when tested at 1 mM concentration.

Inactivation of histidine ammonia-lyase from Streptomyces griseus by dicarbonyl reagents.

Histidine ammonia-lyase from Streptomyces griseus was inactivated by methylglyoxal and phenylglyoxal, dicarbonyl reagents known to react specifically with arginyl residues in proteins. The inactivation showed pseudo-first-order kinetics and could be prevented by protection with histidinol phosphate, a competitive inhibitor of histidine ammonia-lyase. Analysis of the amino acid composition of histidine ammonia-lyase after treatment with phenylglyoxal, together with the kinetics of inactivation, suggested that inactivation was a consequence of specific reaction with one or more essential arginyl residues at or near the active site of the enzyme.

[Biological effect of Botran-75B]. / O biologicheskom deistvii botrana-75B.

Study results on acute and chronic toxicity allowed us to attribute bortran-75B fungicide to low-toxic compounds with moderately expressed cumulation. Under long-term pesticide exposure changes in activity of a number of blood enzymes were noted. Its threshold and nonactive doses were determined.

Reduced locomotor activity of rats made histidinemic by injection of histidine.

Acute histidinemia was provoked in 30-d-old male Wistar rats by injecting intraperitoneally either histidine alone (0.5 mg/g body wt) or histidine (0.25 mg/g body wt) plus the histidase inhibitor nitromethane (0.73 mg/g body wt). Histidase activity was approximately 90% inhibited in rats receiving nitromethane. Serum histidine in both groups reached levels similar to those of histidinemic patients. Rats were subjected to the open field behavioral task, and the number of rearings and crossings were counted. A consistently lower locomotor activity was observed in the histidinemic rats. It is proposed that reduced locomotor activity and its relationship to psychomotor development should be investigated in histidinemic children.

Effect of Zajdela ascites hepatoma on the activity and synthesis of liver histidase of tumor-bearing rats.

The synthesis of histidase occurs only in free polyribosomes. The relative content of histidase synthesizing polyribosomes in rat liver, in Zajdela ascites hepatoma cells and in the liver of tumor-bearing rats is equal to 1.35%, 0.11% and 0.57%, respectively (of the total amount of free polyribosomes). It was found that hepatoma cell sap has an inhibitory effect on the synthesis of proteins in the cell-free system reconstructed from polyribosomes and cell sap of control rats.

Immunological relatedness of histidine ammonia-lyases from some species of Pseudomonas: taxonomic implication.

1. Histidine ammonia-lyases (histidase EC 4.3.1.3) from Pseudomonas testosteroni NCIB 10808 and Pseudomonas putida NCIB 10807 were purified and specific antibody was raised to each separately in a rabbit. 2. Immunological cross-reactions of each antibody to histidine ammonia-lyases from various species of Pseudomonas were examined by the enzyme inhibition test. 3. The immunological data obtained suggest that these Pseudomonas species can be classified into three groups. These cross-reactions tend to indicate a certain degree of homology within species in a group but not between groups.

[Presence of histidine-degrading enzymes in mycobacteria and nocardia and reaction-kinetic studies on Mycobacterium smegmatis SN 2 (author's transl)]. / Verbreitung histidinabbauender Enzyme in Mycobakterien und Nocardien und reaktionskinetische Untersuchungen an Mycobacterium smegmatis SN 2.

The occurrence of histidine degrading enzymes in whole cells of mycobacteria and nocardia was investigated. Out of 25 Mycobacterium strains, only M. smegmatis showed "histidase" activity, i.e. an uptake of histidine and a simultaneous release of ammonia. Consequently, the presence of "histidase" is a characteristic feature for M. smegmatis. In contrast to "histidase", single mycobacteria strains were able to take up histidine from the reaction mixture without liberation of ammonia or detectable oxidation of the substrate. This property was not species-specific. Therefore the determination of the released ammonia is decisive for the determination of the species-specific "histidase". Strains of the genus Nocardia proved to be more active concerning "histidase". Out of 18 species we found activity in all 4 N. erythropolis strains and in 7 out of 10 N. asteroides strains; the only strains of the species N. restrictus, N. opaca, N. blackwellii, N. paraffinae and R. terrae also showed "histidase" activity. Under the experimental conditions whereby 1 mmol/l of histidine and 10 mg/ml of bacteria were used, histidine was degraded by M. smegmatis within 6--8 hours; during which reaction 1,7--1,8 mmole/l of ammonia was released and 5/2--6/2 mmol of oxygen consumed. Other metabolites, such as urocanic acid or glutamic acid were not released in the reaction mixture. The kinetics of the degradation by whole cells was investigated in detail; the Michaelis constant was 0,53 . 10(-3)M, the optimal temperature found at 43,2 degree C, below and above which temperature the activation energies were 5177 and 11806 cal, respectively. "Histidase" is inducible in M. smegmatis. After cultivation of the bacteria on media containing histidine or urocanic acid, the enzymatic activity strongly increased. The same effect could be obtained by preincubating washed cells in buffer containing histidine or urocanic acif for 4 hours. "Histidase" was inhibited by streptomycin, viomycin and p-chlormercuribenzoate. The degeneration pathway is supposed to start from histidine via urocanic acid and glutamic acid; thus the proposed pathway is different from that one suggested by Bönicke (1964). Details of the degradation pathway using partially purified enzyme preparations will be described latter.

L-Histidine ammonia-lyase activity of axenically grown Hartmannella culbertsoni.

1. Histidine ammonia-lyase (EC 4.3.1.3) activity in the cell-free extracts of Hartmannella culbertsoni has been partially purified and the optimum activity is found at pH 9.0--9.2. 2. The enzyme required sulphydryl groups for its activity. L-2-Thiohistidine and EDTA competitively inhibit the enzyme. 3. Its molecular weight, as determined by gel filtration technique, is 131,800 daltons and the energy of activation for this enzyme is 15,205 cals/mole. 4. Certain amoebicidal drug and divalent cations have marked inhibitory effect on the enzyme. Co2+ has a profound stimulatory effect.

Studies on the production and assessment of experimental histidinemia in the rat.

Intraperitoneal administration to rats of D- or DL-alpha-hydrazinoimidazolylpropionic acid was found to produce a substantial inactivation of hepatic histidine ammonia-lyase (EC 4.3.1.3) in vivo. Proportional to this loss in enzyme activity was an impairment of the ability of treated rats to oxidize L-[ring-2-14C] histidine to 14CO2. Rats in which hepatic histidine ammonia-lyase activity was either depressed by DL-hydrazinoimidazolylproprionic acid injection or elevated by feeding a high protein diet displayed proportionately altered rates of 3H2O release into plasma water following L-[3-3H] histidine administration. Plasma L-histidine clearance following loading with this amino acid was similarly affected by these treatments. Administration of DL-alphal-hydrazinoimisazolylproprionic acid to rats was also found to inactivate non-specifically pyridoxal 5-phosphate enzymes in vivo; pyridoxine injection was found to reverse the DL-alpha-hydrazinoimidazolylproprionic acid-induced inactivation of hepatic aspartate aminotransferase (EC 2.6.1.1) in vivo, but not that of hepatic histidine ammonia-lyase. These findings demonstrate that histidine ammonia-lyase is the rate-limiting factor in L-histidine degradation in the rat. The potential usefulness of DL-hydrazinoimidazolylproprionic acid in the production of an animal model for histidinemia (hereditary histidine ammonia-lyase deficiency) is discussed.