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Jan N. M. Commandeur - One of the best experts on this subject based on the ideXlab platform.
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Bioactivation of Chemopreventive Selenocysteine Se-Conjugates and Related Amino Acids by Amino Acid Oxidases Novel Route of Metabolism of Selenoamino Acids
Chemical Research in Toxicology, 2001Co-Authors: Martijn Rooseboom, Nico P. E. Vermeulen, N. Van Hemert, Jan N. M. CommandeurAbstract:Several selenocysteine Se-conjugates have been shown to possess potent chemopreventive activity in animal models for chemical carcinogenesis. As a mechanism of action, Beta-Elimination reactions to form chemopreventive selenols, ammonia, and pyruvate has been proposed. The enzymes involved in these Beta-Elimination reactions, however, have been partially elucidated. Next to cysteine conjugate beta-lyases, as yet unidentified non-pyridoxal-5'-phosphate-dependent enzymes also appear to be involved in cytosolic Beta-Elimination reactions. In the present study, it was investigated whether amino acid oxidases contribute to the bioactivation of selenocysteine Se-conjugates. Using purified L-amino acid oxidase from Crotalus adamanteus as a model enzyme, significant Beta-Elimination activities were indeed observed upon incubation with Se-methylselenocysteine (K(m), 195 microM; k(cat), 48 min(-1)), Se-allylselenocysteine (K(m), 608 microM; k(cat), 34 min(-1)), Se-phenylselenocysteine (K(m), 107 microM; k(cat), 57 min(-1)) and Se-benzylselenocysteine (K(m), 59 microM; k(cat), 13 min(-1)). For all selenocysteine Se-conjugates tested, the rate of pyruvate formation was comparable to that of hydrogen peroxide, one of the products of oxidative deamination. The fact that addition of catalase did not alter pyruvate formation indicated that the Beta-Elimination reaction observed was not mediated by selenoxidation/syn-elimination due to the hydrogen peroxide formed via the oxidative deamination pathway. Using D-amino acid oxidase from porcine kidney and D-SeCys conjugates similar results were obtained. To delineate whether mammalian L-amino acid oxidases are also able to catalyze Beta-Elimination of selenocysteine Se-conjugates, rat renal cytosol was fractionated and screened for Beta-Elimination and oxidative deamination activities. One of the fractions isolated displayed oxidative deamination activity with several amino acids and cysteine S-conjugates. With selenocysteine Se-conjugates as substrates, however, this fraction displayed both oxidative deamination and Beta-Elimination activities, when incubated in the presence of aminoxyacetic acid to block contribution of pyridoxal-5'-phosphate-dependent enzymes. The potential significance of this novel bioactivation route for the chemopreventive activity of selenocysteine Se-conjugates is discussed.
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bioactivation of selenocysteine se conjugates by a highly purified rat renal cysteine conjugate beta lyase glutamine transaminase k
Journal of Pharmacology and Experimental Therapeutics, 2000Co-Authors: Jan N. M. Commandeur, Ioanna Andreadou, Martijn Rooseboom, M Out, L J De Leur, E J Groot, Nico P. E. VermeulenAbstract:Selenocysteine Se-conjugates have recently been proposed as potential prodrugs to target pharmacologically active selenol compounds to the kidney. Although rat renal cytosol displayed a high activity of Beta-Elimination activity toward these substrates, the enzymes involved in this activation pathway as yet have not been identified. In the present study, the possible involvement of cysteine conjugate beta-lyase/glutamine transaminase K (beta-lyase/GTK) in cytosolic activity was investigated. To this end, the enzyme kinetics of 15 differentially substituted selenocysteine Se-conjugates and 11 cysteine S-conjugates was determined using highly purified rat renal beta-lyase/GTK. The results demonstrate that most selenocysteine Se-conjugates are beta-eliminated at a very high activity by purified beta-lyase/GTK, implicating an important role of this protein in the previously reported Beta-Elimination reactions in rat renal cytosol. As indicated by the rapid consumption of alpha-keto-gamma-methiolbutyric acid, purified beta-lyase/GTK also catalyzed transamination reactions, which appeared to even exceed that of Beta-Elimination. The corresponding sulfur analogs also showed significant transamination but were beta-eliminated at an extremely low rate. Comparison of the obtained enzyme kinetic data of purified beta-lyase/GTK with previously obtained data from rat renal cytosol showed a poor correlation. By determining the activity profiles of cytosolic fractions applied to anion exchange fast protein liquid chromatography and gel filtration chromatography, the involvement of multiple enzymes in the Beta-Elimination of selenocysteine Se-conjugates in rat renal cytosol was demonstrated. The identity and characteristics of these alternative selenocysteine conjugate beta-lyases, however, remain to be established.
Nico P. E. Vermeulen - One of the best experts on this subject based on the ideXlab platform.
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Bioactivation of Chemopreventive Selenocysteine Se-Conjugates and Related Amino Acids by Amino Acid Oxidases Novel Route of Metabolism of Selenoamino Acids
Chemical Research in Toxicology, 2001Co-Authors: Martijn Rooseboom, Nico P. E. Vermeulen, N. Van Hemert, Jan N. M. CommandeurAbstract:Several selenocysteine Se-conjugates have been shown to possess potent chemopreventive activity in animal models for chemical carcinogenesis. As a mechanism of action, Beta-Elimination reactions to form chemopreventive selenols, ammonia, and pyruvate has been proposed. The enzymes involved in these Beta-Elimination reactions, however, have been partially elucidated. Next to cysteine conjugate beta-lyases, as yet unidentified non-pyridoxal-5'-phosphate-dependent enzymes also appear to be involved in cytosolic Beta-Elimination reactions. In the present study, it was investigated whether amino acid oxidases contribute to the bioactivation of selenocysteine Se-conjugates. Using purified L-amino acid oxidase from Crotalus adamanteus as a model enzyme, significant Beta-Elimination activities were indeed observed upon incubation with Se-methylselenocysteine (K(m), 195 microM; k(cat), 48 min(-1)), Se-allylselenocysteine (K(m), 608 microM; k(cat), 34 min(-1)), Se-phenylselenocysteine (K(m), 107 microM; k(cat), 57 min(-1)) and Se-benzylselenocysteine (K(m), 59 microM; k(cat), 13 min(-1)). For all selenocysteine Se-conjugates tested, the rate of pyruvate formation was comparable to that of hydrogen peroxide, one of the products of oxidative deamination. The fact that addition of catalase did not alter pyruvate formation indicated that the Beta-Elimination reaction observed was not mediated by selenoxidation/syn-elimination due to the hydrogen peroxide formed via the oxidative deamination pathway. Using D-amino acid oxidase from porcine kidney and D-SeCys conjugates similar results were obtained. To delineate whether mammalian L-amino acid oxidases are also able to catalyze Beta-Elimination of selenocysteine Se-conjugates, rat renal cytosol was fractionated and screened for Beta-Elimination and oxidative deamination activities. One of the fractions isolated displayed oxidative deamination activity with several amino acids and cysteine S-conjugates. With selenocysteine Se-conjugates as substrates, however, this fraction displayed both oxidative deamination and Beta-Elimination activities, when incubated in the presence of aminoxyacetic acid to block contribution of pyridoxal-5'-phosphate-dependent enzymes. The potential significance of this novel bioactivation route for the chemopreventive activity of selenocysteine Se-conjugates is discussed.
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bioactivation of selenocysteine se conjugates by a highly purified rat renal cysteine conjugate beta lyase glutamine transaminase k
Journal of Pharmacology and Experimental Therapeutics, 2000Co-Authors: Jan N. M. Commandeur, Ioanna Andreadou, Martijn Rooseboom, M Out, L J De Leur, E J Groot, Nico P. E. VermeulenAbstract:Selenocysteine Se-conjugates have recently been proposed as potential prodrugs to target pharmacologically active selenol compounds to the kidney. Although rat renal cytosol displayed a high activity of Beta-Elimination activity toward these substrates, the enzymes involved in this activation pathway as yet have not been identified. In the present study, the possible involvement of cysteine conjugate beta-lyase/glutamine transaminase K (beta-lyase/GTK) in cytosolic activity was investigated. To this end, the enzyme kinetics of 15 differentially substituted selenocysteine Se-conjugates and 11 cysteine S-conjugates was determined using highly purified rat renal beta-lyase/GTK. The results demonstrate that most selenocysteine Se-conjugates are beta-eliminated at a very high activity by purified beta-lyase/GTK, implicating an important role of this protein in the previously reported Beta-Elimination reactions in rat renal cytosol. As indicated by the rapid consumption of alpha-keto-gamma-methiolbutyric acid, purified beta-lyase/GTK also catalyzed transamination reactions, which appeared to even exceed that of Beta-Elimination. The corresponding sulfur analogs also showed significant transamination but were beta-eliminated at an extremely low rate. Comparison of the obtained enzyme kinetic data of purified beta-lyase/GTK with previously obtained data from rat renal cytosol showed a poor correlation. By determining the activity profiles of cytosolic fractions applied to anion exchange fast protein liquid chromatography and gel filtration chromatography, the involvement of multiple enzymes in the Beta-Elimination of selenocysteine Se-conjugates in rat renal cytosol was demonstrated. The identity and characteristics of these alternative selenocysteine conjugate beta-lyases, however, remain to be established.
Martijn Rooseboom - One of the best experts on this subject based on the ideXlab platform.
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Bioactivation of Chemopreventive Selenocysteine Se-Conjugates and Related Amino Acids by Amino Acid Oxidases Novel Route of Metabolism of Selenoamino Acids
Chemical Research in Toxicology, 2001Co-Authors: Martijn Rooseboom, Nico P. E. Vermeulen, N. Van Hemert, Jan N. M. CommandeurAbstract:Several selenocysteine Se-conjugates have been shown to possess potent chemopreventive activity in animal models for chemical carcinogenesis. As a mechanism of action, Beta-Elimination reactions to form chemopreventive selenols, ammonia, and pyruvate has been proposed. The enzymes involved in these Beta-Elimination reactions, however, have been partially elucidated. Next to cysteine conjugate beta-lyases, as yet unidentified non-pyridoxal-5'-phosphate-dependent enzymes also appear to be involved in cytosolic Beta-Elimination reactions. In the present study, it was investigated whether amino acid oxidases contribute to the bioactivation of selenocysteine Se-conjugates. Using purified L-amino acid oxidase from Crotalus adamanteus as a model enzyme, significant Beta-Elimination activities were indeed observed upon incubation with Se-methylselenocysteine (K(m), 195 microM; k(cat), 48 min(-1)), Se-allylselenocysteine (K(m), 608 microM; k(cat), 34 min(-1)), Se-phenylselenocysteine (K(m), 107 microM; k(cat), 57 min(-1)) and Se-benzylselenocysteine (K(m), 59 microM; k(cat), 13 min(-1)). For all selenocysteine Se-conjugates tested, the rate of pyruvate formation was comparable to that of hydrogen peroxide, one of the products of oxidative deamination. The fact that addition of catalase did not alter pyruvate formation indicated that the Beta-Elimination reaction observed was not mediated by selenoxidation/syn-elimination due to the hydrogen peroxide formed via the oxidative deamination pathway. Using D-amino acid oxidase from porcine kidney and D-SeCys conjugates similar results were obtained. To delineate whether mammalian L-amino acid oxidases are also able to catalyze Beta-Elimination of selenocysteine Se-conjugates, rat renal cytosol was fractionated and screened for Beta-Elimination and oxidative deamination activities. One of the fractions isolated displayed oxidative deamination activity with several amino acids and cysteine S-conjugates. With selenocysteine Se-conjugates as substrates, however, this fraction displayed both oxidative deamination and Beta-Elimination activities, when incubated in the presence of aminoxyacetic acid to block contribution of pyridoxal-5'-phosphate-dependent enzymes. The potential significance of this novel bioactivation route for the chemopreventive activity of selenocysteine Se-conjugates is discussed.
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bioactivation of selenocysteine se conjugates by a highly purified rat renal cysteine conjugate beta lyase glutamine transaminase k
Journal of Pharmacology and Experimental Therapeutics, 2000Co-Authors: Jan N. M. Commandeur, Ioanna Andreadou, Martijn Rooseboom, M Out, L J De Leur, E J Groot, Nico P. E. VermeulenAbstract:Selenocysteine Se-conjugates have recently been proposed as potential prodrugs to target pharmacologically active selenol compounds to the kidney. Although rat renal cytosol displayed a high activity of Beta-Elimination activity toward these substrates, the enzymes involved in this activation pathway as yet have not been identified. In the present study, the possible involvement of cysteine conjugate beta-lyase/glutamine transaminase K (beta-lyase/GTK) in cytosolic activity was investigated. To this end, the enzyme kinetics of 15 differentially substituted selenocysteine Se-conjugates and 11 cysteine S-conjugates was determined using highly purified rat renal beta-lyase/GTK. The results demonstrate that most selenocysteine Se-conjugates are beta-eliminated at a very high activity by purified beta-lyase/GTK, implicating an important role of this protein in the previously reported Beta-Elimination reactions in rat renal cytosol. As indicated by the rapid consumption of alpha-keto-gamma-methiolbutyric acid, purified beta-lyase/GTK also catalyzed transamination reactions, which appeared to even exceed that of Beta-Elimination. The corresponding sulfur analogs also showed significant transamination but were beta-eliminated at an extremely low rate. Comparison of the obtained enzyme kinetic data of purified beta-lyase/GTK with previously obtained data from rat renal cytosol showed a poor correlation. By determining the activity profiles of cytosolic fractions applied to anion exchange fast protein liquid chromatography and gel filtration chromatography, the involvement of multiple enzymes in the Beta-Elimination of selenocysteine Se-conjugates in rat renal cytosol was demonstrated. The identity and characteristics of these alternative selenocysteine conjugate beta-lyases, however, remain to be established.
Thuong B. Nguyen - One of the best experts on this subject based on the ideXlab platform.
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Use of 2,6-dihydroxyacetophenone for analysis of fragile peptides, disulphide bonding and small proteins by matrix-assisted laser desorption/ionization.
Rapid communications in mass spectrometry : RCM, 1996Co-Authors: Jeffrey J. Gorman, Bettina L. Ferguson, Thuong B. NguyenAbstract:Several peptides were shown to undergo fragmentation during matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to a degree which complicated their analysis using alpha-cyano-4-hydroxycinnamic acid (CHCA) as a matrix, even at threshold laser irradiance. These peptides included synthetic peptides, peptides isolated from viral proteins and a phosphopeptide from beta-casein (residues 33-48). The excessive fragmentation occurred usually as a post-source phenomenon; however, in-source fragmentation was also observed. The combined effects of in-source and post-source fragmentation of one peptide studied led to a failure to observe the protonated molecule of this peptide in reflector mode analysis. The phosphopeptide studied exhibited a high degree of Beta-Elimination of phosphate. It was demonstrated that the fragility exhibited by these peptides in CHCA, including Beta-Elimination of phosphate from serine, was not evident with a matrix comprising 2,6-dihydroxyacetophenone (DHAP) and di-ammonium hydrogen citrate (DAHC). The DHAP/DAHC matrix was also adapted for direct analysis of peptides from an acidic reducing milieu containing tris(2-carboxyethyl)phosphine. The molecular weight of equine cytochrome c was determined with a relatively high degree of accuracy (experimental M(r) = 12360.2 +/- 1.4 Da compared to the theoretical M(r) = 12360.09 Da) using DHAP/DAHC as a matrix for reflector mode analysis.
Philipp Christen - One of the best experts on this subject based on the ideXlab platform.
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conversion of tyrosine phenol lyase to dicarboxylic amino acid beta lyase an enzyme not found in nature
Journal of Biological Chemistry, 1999Co-Authors: Barbara Mouratou, Patrik Kasper, Heinz Gehring, Philipp ChristenAbstract:Tyrosine phenol-lyase (TPL), which catalyzes the Beta-Elimination reaction of L-tyrosine, and aspartate aminotransferase (AspAT), which catalyzes the reversible transfer of an amino group from dicarboxylic amino acids to oxo acids, both belong to the alpha-family of vitamin B6-dependent enzymes. To switch the substrate specificity of TPL from L-tyrosine to dicarboxylic amino acids, two amino acid residues of AspAT, thought to be important for the recognition of dicarboxylic substrates, were grafted into the active site of TPL. Homology modeling and molecular dynamics identified Val-283 in TPL to match Arg-292 in AspAT, which binds the distal carboxylate group of substrates and is conserved among all known AspATs. Arg-100 in TPL was found to correspond to Thr-109 in AspAT, which interacts with the phosphate group of the coenzyme. The double mutation R100T/V283R of TPL increased the Beta-Elimination activity toward dicarboxylic amino acids at least 10(4)-fold. Dicarboxylic amino acids (L-aspartate, L-glutamate, and L-2-aminoadipate) were degraded to pyruvate, ammonia, and the respective monocarboxylic acids, e.g. formate in the case of L-aspartate. The activity toward L-aspartate (kcat = 0.21 s-1) was two times higher than that toward L-tyrosine. Beta-Elimination and transamination as a minor side reaction (kcat = 0.001 s-1) were the only reactions observed. Thus, TPL R100T/V283R accepts dicarboxylic amino acids as substrates without significant change in its reaction specificity. Dicarboxylic amino acid beta-lyase is an enzyme not found in nature.
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evolutionary relationships among pyridoxal 5 phosphate dependent enzymes
FEBS Journal, 1994Co-Authors: Frederick W. Alexander, Erika Sandmeier, Perdeep K. Mehta, Philipp ChristenAbstract:Pyridoxal-5'-phosphate-dependent enzymes catalyze manifold reactions in the metabolism of amino acids. A comprehensive comparison of amino acid sequences has shown that most of these enzymes can be assigned to one of three different families of homologous proteins. The sequences of the enzymes of each family were aligned and their homology confirmed by profile analysis. Scrutiny of the reactions catalyzed by the enzymes showed that their affiliation with one of the three structurally defined families correlates in most cases with their regio-specificity. In the largest family, the covalency changes of the substrate occur at the same carbon atom that carries the amino group forming the imine linkage with the coenzyme. This family was thus named alpha family. It comprises glycine hydroxymethyltransferase, glycine C-acetyltransferase, 5-aminolevulinate synthase, 8-amino-7-oxononanoate synthase, all aminotransferases (with the possible exception of subgroup III), a number of other enzymes relatively closely related with the aminotransferases and very likely a certain group of amino acid decarboxylases as well as tryptophanase and tyrosine phenol-lyase which, however, catalyze Beta-Elimination reactions. The beta family includes L- and D-serine dehydratase, threonine dehydratase, the beta subunit of tryptophan synthase, threonine synthase and cysteine synthase. These enzymes catalyze beta-replacement or Beta-Elimination reactions. The gamma family incorporates O-succinylhomoserine (thiol-lyase, O-acetylhomoserine (thiol)-lyase, and cystathionine gamma-lyase, which catalyze gamma-replacement or gamma-elimination reactions, as well as cystathionine beta-lyase. The alpha and gamma family might be distantly related with one another, but are clearly not homologous with the beta family. Apparently, the primordial pyridoxal-5'-phosphate-dependent enzymes were regio-specific catalysts, which first specialized for reaction specificity and then for substrate specificity. The following pyridoxal-5'-phosphate-dependent enzymes seem to be unrelated with the alpha, beta or gamma family by the criterion of profile analysis:alanine racemase, selenocysteine synthase, and many amino acid decarboxylases. These enzymes may represent yet other families of B6 enzymes.