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PubMed2003

Production of cysteine for bacterial and plant biotechnology: application of cysteine feedback-insensitive isoforms of serine acetyltransferase.

The first step of cysteine biosynthesis in bacteria and plants consists in the formation of O-acetylserine catalyzed by serine acetyltransferase (SAT). SAT is highly sensitive to feedback inhibition by cysteine as part of the regulatory circuit of cysteine biosynthesis und thus hampers over-expression and fermentation of cysteine in biotechnological production processes. Since plants contain multiple SAT isoforms with different cysteine feedback sensitivity, this resource was exploited to demonstrate the suitability of plant SATs for the production of cysteine in both bacteria and plants. Three new cDNAs encoding SATs were isolated from Nicotiana tabacum. The catalytic activity of SAT4 was insensitive up to 0.6 mM cysteine. Expression of SAT4 in a newly constructed Escherichia coli host strain without endogenous SAT activity yielded a significant accumulation of cysteine in the culture medium compared to expression of cysteine sensitive SATs in the same strain. The application of a similarly insensitive SAT isoform from A. thaliana demonstrated the suitability of this approach to increase cysteine levels in transgenic tobacco plants.

Acetyltransferases↗

PubMed1991

Modulation of lymphocyte functions and immune responses by cysteine and cysteine derivatives.

Mitogenically stimulated human peripheral blood lymphocytes and T cell clones were found to have weak membrane transport activity for the disulfide cystine but strong membrane transport activity for the thiol amino acid cysteine. Cysteine, however, is represented at the lowest concentration among all protein-forming amino acids in the blood plasma. Complementary laboratory experiments have shown that the cysteine supply is indeed limiting for important lymphocyte functions. Proliferative responses of mitogenically stimulated lymphocytes and T-cell clones and the activation of cytotoxic T cells in allogeneic mixed lymphocyte cultures are strongly influenced by small variations in the extracellular cysteine concentration even in the presence of relatively high and approximately physiologic concentrations of cystine. Cysteine can be substituted by N-acetylcysteine but not by cystine. The more detailed analysis revealed that the extracellular supply of cysteine influences strongly the intracellular level of glutathione (GSH) and also the activity of the transcription factor NF kappa B that regulates the expression of several immunologically relevant genes. In vitro experiments including double-chamber experiments with macrophages and lymphocytes revealed, moreover, that cysteine plays an important role as a regulatory mediator between these cell types. The cysteine supply is impaired directly or indirectly in several pathologic conditions that are associated with immunodeficiencies, including the acquired immune deficiency syndrome (AIDS). Cysteine or cysteine derivatives may therefore be considered for the treatment of patients with HIV-1 infection.

Animals↗

PubMed1988

Bacterial cysteine conjugate beta-lyase and the metabolism of cysteine S-conjugates: structural requirements for the cleavage of S-conjugates and the formation of reactive intermediates.

The cysteine conjugate beta-lyase mediated metabolism and the mutagenicity of the synthetic cysteine conjugates S-(2-chloroethyl)-L-cysteine (CEC), S-(2-chlorovinyl)-L-cysteine (CVC), S-(1,2,3,3,3-pentachloroprop-1-enyl)-L-cysteine (PCPC), S-(pentachlorophenyl)-L-cysteine (PCPhC), S-(chloro-1,2,2-trifluoroethyl)-L-cysteine (CTFEC), S-benzyl-L-cysteine (SBC) and S-methyl-L-cysteine (SMC) were investigated in Salmonella typhimurium strains TA100, TA2638, TA102 and TA98 to establish structure/activity relationships. Bacterial 100,000 X g supernatants cleaved CTFEC, PCPC, CVC, PCPhC and SBC to pyruvate; pyruvate formation was inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA) in all cases. Of the compounds tested, CEC, PCPC and CVC were mutagenic in the Ames-test. CTFEC, PCPhC and SBC failed to increase the number of revertants above control levels. The mutagenicity of PCPC and CVC could be inhibited by AOAA. CEC exerted a potent mutagenic effect in the Ames-test which was not affected by AOAA; CEC was not transformed to pyruvate by bacterial beta-lyase. Neither pyruvate formation nor mutagenicity were observed with SMC. These results indicate that the structure of the substituent on the sulfur atom is an important determinant for the biological activity of cysteine S-conjugates. Electronegative and/or unsaturated substituents are required for beta-lyase catalysed beta-elimination reactions. The formation of chemically unstable thiols, which may be converted to thioacylating intermediates, seems to be a prerequisite for beta-lyase dependent mutagenicity of S-conjugates.

Aminooxyacetic Acid↗

PubMed1986

Characterization of two cysteine residues in cytochrome P-450scc: chemical identification of the heme-binding cysteine residue.

It was found that there were only two cysteine residues in highly purified cytochrome P-450scc molecule from bovine adrenocortical mitochondria by titration with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) in denatured conditions. Only one cysteine residue at position 303 of cytochrome P-450scc could be specifically modified with DTNB in the native state. The resulting cytochrome P-450scc-5-thio-2-nitrobenzoic acid complex (cytochrome P-450scc-TNB) showed no distinct differences in absorption spectra, cholesterol binding, or electron transferring from adrenodoxin, compared to those of untreated cytochrome P-450scc. These observations indicated that the 303rd cysteine residue does not play a role in heme binding, cholesterol (substrate) binding or adrenodoxin binding. The other cysteine residue at 461 could be modified with DTNB only in a denatured condition. These assignments of cysteine residues were made by the subsequent S-cyanylation with KCN followed by incubation in 6 M guanidine hydrochloride at alkaline pH, which causes enhanced cleavage of peptide bonds adjacent to the cyanylated cysteine residues. Analyses of fragmented polypeptides by SDS-polyacrylamide gel electrophoresis confirmed that there were only two cysteine residues in the molecule and indicated that the cleavage rate of the peptide bond between 460 and 461 becomes high only when both cysteine residues (303 and 461) are cyanylated. These results clearly established that the 461st cysteine residue in cytochrome P-450scc plays a role as the heme fifth ligand on the basis of the general agreement that a thiolated cysteine residue coordinates to the heme iron.

Adrenal Cortex↗

PubMed2004

Regulation of cysteine dioxygenase and gamma-glutamylcysteine synthetase is associated with hepatic cysteine level.

Two hepatic enzymes, cysteine dioxygenase (CDO) and gamma-glutamylcysteine synthetase (GCS), play important regulatory roles in the response of cysteine metabolism to changes in dietary sulfur amino acid or protein levels. To examine the time-course of changes in CDO and GCS activities, CDO and GCS-catalytic or heavy subunit protein and mRNA levels, and cysteine and glutathione levels, we adapted rats to either a low protein (LP) or high protein (HP) diet, switched them to the opposite diet, and followed these parameters over 6 days. Hepatic CDO activity and amount, but not mRNA level, increased in response to higher protein intake; the t(1/2) of change for CDO activity or protein level was 22 h for rats switched from a LP to a HP diet and 8 h for rats switched from a HP to a LP diet, suggesting that the HP diet decreased turnover of CDO. Hepatic GCS activity, catalytic subunit amount and mRNA level decreased in response to a higher protein intake. GCS catalytic subunit level changed with a similar t(1/2) for both groups, but the change in GCS activity in rats switched from a LP diet to a HP diet was faster (approximately 16h) than for rats switched from a HP to a LP diet (approximately 74h). Hepatic cysteine and glutathione levels reached new steady states within 12 h (LP to HP) or 24 h (HP to LP). CDO activity appeared to be regulated at the level of protein, probably by diminished turnover of CDO in response to higher protein intake or cysteine level, whereas GCS activity appeared to be regulated both at the level of mRNA and activity state in response to the change in cysteine or protein availability. These findings support a role of cysteine concentration as a mediator of its own metabolism, favoring catabolism when cysteine is high and glutathione synthesis when cysteine is low.

Animals↗

PubMed2006

Identification and characterization of bacterial cysteine dioxygenases: a new route of cysteine degradation for eubacteria.

In metazoa and fungi, the catabolic dissimilation of cysteine begins with its sulfoxidation to cysteine sulfinic acid by the enzyme cysteine dioxygenase (CDO). In these organisms, CDO plays an important role in the homeostatic regulation of steady-state cysteine levels and provides important oxidized metabolites of cysteine such as sulfate and taurine. To date, there has been no experimental evidence for the presence of CDO in prokaryotes. Using PSI-BLAST searches and crystallographic information about the active-site geometry of mammalian CDOs, we identified a total of four proteins from Bacillus subtilis, Bacillus cereus, and Streptomyces coelicolor A3(2) that shared low overall identity to CDO (13 to 21%) but nevertheless conserved important active-site residues. These four proteins were heterologously expressed and purified to homogeneity by a single-step immobilized metal affinity chromatography procedure. The ability of these proteins to oxidize cysteine to cysteine sulfinic acid was then compared against recombinant rat CDO. The kinetic data strongly indicate that these proteins are indeed bona fide CDOs. Phylogenetic analyses of putative bacterial CDO homologs also indicate that CDO is distributed among species within the phyla of Actinobacteria, Firmicutes, and Proteobacteria. Collectively, these data suggest that a large subset of eubacteria is capable of cysteine sulfoxidation. Suggestions are made for how this novel pathway of cysteine metabolism may play a role in the life cycle of the eubacteria that have it.

Bacteria↗

PubMed2002

Novel cysteine proteinase inhibitors homologous to the proregions of cysteine proteinases.

Propeptides of papain-like cysteine proteinases such as papain, cathepsins B, L and S are potent inhibitors of their cognate cysteine proteinases with Ki values in the nanomolar range, and they exhibit highest inhibition selectivity for enzymes from which they originate. Recent studies have identified novel inhibitor proteins that are homologous to the proregions of papain-like cysteine proteinases. Mouse activated T-lymphocytes express cytotoxic T-lymphocyte antigen (CTLA-2), which is homologous to the proregion of mouse cathepsin L. CTLA-2 exhibits inhibitory activities to several cysteine proteinases. We have also identified a similar propeptide-like cysteine proteinase inhibitor, Bombyx cysteine proteinase inhibitor (BCPI), in the silkmoth Bombyx mori. BCPI is a slow and tight binding inhibitor of cathepsin L-like cysteine proteinases with Ki values in picomolar range, and the inhibition is highly selective towards these proteinases just like the propeptides. Recent genome analyses have shown the expression of similar propeptide-like proteins in Drosophila and rat, suggesting the presence of a novel class of cysteine proteinase inhibitors in a variety of organisms. Studies of the gene structures and phylogenetic analysis have shown that genes of the propeptide-like cysteine proteinase inhibitors have emerged from ancestor genes of their parental enzymes.

Amino Acid Sequence↗

PubMed2005

L-cysteine desulfidase: an [4Fe-4S] enzyme isolated from Methanocaldococcus jannaschii that catalyzes the breakdown of L-cysteine into pyruvate, ammonia, and sulfide.

A [4Fe-4S] enzyme that decomposes L-cysteine to hydrogen sulfide, ammonia, and pyruvate has been isolated and characterized from Methanocaldococcus jannaschii. The sequence of the isolated enzyme demonstrated that the protein was the product of the M. jannaschii MJ1025 gene. The protein product of this gene was recombinantly produced in Escherichia coli and purified to homogeneity. Both the isolated and recombinant enzymes are devoid of pyridoxal phosphate (PLP) and are rapidly inactivated upon exposure to air. The air-inactivated enzyme is activated by reaction with Fe2+ and dithiothreitol in the absence of air. The air-inactivated enzyme contains 3 mol of iron per subunit (43 kDa, SDS gel electrophoresis), and the native enzyme has a measured molecular mass of 135 kDa (gel filtration), indicating it is a trimer. The enzyme is very specific for L-cysteine, with no activity being detected with D-cysteine, L-homocysteine, 3-mercaptopropionic acid (cysteine without the amino group), cysteamine (cysteine without the carboxylic acid), or mercaptolactate (the hydroxyl analogue of cysteine). The activity of the enzyme was stimulated by 40% when the enzyme was assayed in the presence of methyl viologen (4 mM) and inhibited by 70% when the enzyme was assayed in the presence of EDTA (7.1 mM). Preincubation of the enzyme with iodoacetamide (17 mM) completely abolishes activity. The enzymatic activity has a half-life of 8 or 12 min when the enzyme is treated at room temperature with 0.42 mM N-ethylmaleimide (NEM) or 0.42 mM iodoacetamide, respectively. MALDI analysis of the NEM-inactivated enzyme showed Cys25 as the site of alkylation. Site-directed mutagenesis of each of four of the cysteines conserved in the orthologues of the enzyme reduced the catalytic efficiency and thermal stability of the enzyme. The enzyme was found to catalyze exchange of the C-2 hydrogen of the L-cysteine with solvent. These results are consistent with three of the conserved cysteines being involved in the formation of the [4Fe-4S] center and the thiolate of Cys25 serving as a base to abstract the alpha-hydrogen in the first step of the elimination. Although the enzyme has no sequence homology to any known enzymes, including the non-PLP-dependent serine/threonine dehydratases or aconitases, the mechanisms of action of all of these enzymes are similar, in that each catalyzes an alpha,beta-elimination reaction adjacent to a carboxylate group. It is proposed that the enzyme may be responsible for the production of sulfide required for the biosynthesis of iron-sulfur centers in this archaea. A mechanism of action of the enzyme is proposed.

Ammonia↗

PubMed1999

Characterization of the gene encoding serine acetyltransferase, a regulated enzyme of cysteine biosynthesis from the protist parasites Entamoeba histolytica and Entamoeba dispar. Regulation and possible function of the cysteine biosynthetic pathway in Entamoeba.

The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36-52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by L-cystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.

Acetyltransferases↗

PubMed2006

Crystal structure of mammalian cysteine dioxygenase. A novel mononuclear iron center for cysteine thiol oxidation.

Cysteine dioxygenase is a mononuclear iron-dependent enzyme responsible for the oxidation of cysteine with molecular oxygen to form cysteine sulfinate. This reaction commits cysteine to either catabolism to sulfate and pyruvate or the taurine biosynthetic pathway. Cysteine dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat cysteine dioxygenase has been determined to 1.5-A resolution, and these results confirm the canonical cupin beta-sandwich fold and the rare cysteinyltyrosine intramolecular cross-link (between Cys(93) and Tyr(157)) seen in the recently reported murine cysteine dioxygenase structure. In contrast to the catalytically inactive mononuclear Ni(II) metallocenter present in the murine structure, crystallization of a catalytically competent preparation of rat cysteine dioxygenase revealed a novel tetrahedrally coordinated mononuclear iron center involving three histidines (His(86), His(88), and His(140)) and a water molecule. Attempts to acquire a structure with bound ligand using either cocrystallization or soaking crystals with cysteine revealed the formation of a mixed disulfide involving Cys(164) near the active site, which may explain previously observed substrate inhibition. This work provides a framework for understanding the molecular mechanisms involved in thiol dioxygenation and sets the stage for exploration of the chemistry of both the novel mononuclear iron center and the catalytic role of the cysteinyl-tyrosine linkage.

Amino Acid Sequence↗

PubMed1994

Editing function of Escherichia coli cysteinyl-tRNA synthetase: cyclization of cysteine to cysteine thiolactone.

A cyclic sulfur compound, identified as cysteine thiolactone by several chemical and enzymatic tests, is formed from cysteine during in vitro tRNA(Cys) aminoacylation catalyzed by Escherichia coli cysteinyl-tRNA synthetase. The mechanism of cysteine thiolactone formation involves enzymatic deacylation of Cys-tRNA(Cys) (k = 0.017 s-1) in which nucleophilic sulfur of the side chain of cysteine in Cys-tRNA(Cys) attacks its carboxyl carbon to yield cysteine thiolactone. Nonenzymatic deacylation of Cys-tRNA(Cys) (k = 0.0006 s-1) yields cysteine, as expected. Inhibition of enzymatic deacylation of Cys-tRNA(Cys) by cysteine and Cys-AMP, but not by ATP, indicates that both synthesis of Cys-tRNA(Cys) and cyclization of cysteine to the thiolactone occur in a single active site of the enzyme. The cyclization of cysteine is mechanistically similar to the editing reactions of methionyl-tRNA synthetase. However, in contrast to methionyl-tRNA synthetase which needs the editing function to reject misactivated homocysteine, cysteinyl-tRNA synthetase is highly selective and is not faced with a problem in rejecting noncognate amino acids. Despite this, the present day cysteinyl-tRNA synthetase, like methionyl-tRNA synthetase, still retains an editing activity toward the cognate product, the charged tRNA. This function may be a remnant of a chemistry used by an ancestral cysteinyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗

PubMed2002

Biotransformation of L-cysteine S-conjugates and N-acetyl-L-cysteine S-conjugates of the sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (compound A) in human kidney in vitro: interindividual variability in N-acetylation, N-deacetylation, and beta-lyase-catalyzed metabolism.

Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE; 1) is a fluoroalkene formed by the base-catalyzed degradation of the anesthetic sevoflurane. FDVE is nephrotoxic in rats. In both rats and humans, FDVE undergoes glutathione-dependent conjugation, cleavage to cysteine S-conjugates, and renal beta-lyase-catalyzed metabolism to reactive intermediates, which may cause nephrotoxicity. Interindividual variability in renal metabolism of FDVE is unknown. Therefore, this investigation quantified beta-lyase-catalyzed bioactivation and N-acetyltransferase-catalyzed inactivation of FDVE cysteine S-conjugates and reactivation of mercapturates by N-deacetylase in cytosol and microsomes from 20 human kidneys. In cytosol, N-acetylation ranged from 0.008 to 0.045 (0.024 +/- 0.01) nmol of mercapturate/mg/min and 0.001 to 0.07 (0.024 +/- 0.02) nmol of mercapturate/mg/min for alkane and alkene cysteine S-conjugates, respectively. Similar results for microsomal N-acetylation were obtained; N-acetylation ranged from 0.005 to 0.055 (0.025 +/- 0.02) nmol of mercapturate/mg/min and 0.001 to 0.06 (0.030 +/- 0.02) nmol of mercapturate/mg/min for alkane and alkene cysteine S-conjugates, respectively. Beta-lyase-catalyzed metabolism to pyruvate varied from 0.004 to 0.14 (0.051 +/- 0.04) nmol/mg/min and from 0.10 to 0.40 (0.26 +/- 0.08) nmol/mg/min for alkane and alkene cysteine-S-conjugates, respectively. N-deacetylation of mercapturates ranged from 0.8 to 2.5 (1.25 +/- 0.57) nmol of cysteine S-conjugate formed/mg/min and 0.05 to 0.37 (0.17 +/- 0.10) nmol of cysteine S-conjugate formed/mg/min for alkane and alkene FDVE mercapturates. Cytosolic cysteine S-conjugates metabolism by renal beta-lyase predominated over N-acetylation (ratio of activities was 0.2-6 and 3-146 for the alkane and alkene cysteine S-conjugates). N-deacetylation predominated over N-acetylation (ratio of activities was 20-205 and 2-54 for alkane and alkene S-conjugates). There was considerable (up to 50-fold) interindividual variability in rates of FDVE toxication (beta-lyase metabolism and N-deacetylation) and detoxication. This interindividual variability may effect individual susceptibility to the nephrotoxicity of FDVE and other haloalkenes.

Acetylation↗

PubMed1996

Inactivation of brain and kidney aspartate aminotransferases by S-(1,2,-dichlorovinyl)-L-cysteine and by S-(1,1,2,2,-tetrafluoroethyl)-L-cysteine.

Long-term exposure to trichloroethylene can cause kidney cancer in experimental animals and humans. In addition, dichloroacetylene (a breakdown product of trichloroethylene) is nephrotoxic and neurotoxic. Both trichloroethylene and dichloroacetylene are metabolized in part to the corresponding cysteine S-conjugate (i.e. S-(1,2-dichlorovinyl)-L-cysteine) which is toxic. Cysteine S-conjugate beta-lyases convert S-(1,2,dichlorovinyl)-L-cysteine to pyruvate, ammonia and a reactive fragment that adds to macromolecules, depletes cellular thiols and causes lipid peroxidation. We now show that S-(1,2-dichlorovinyl)-L-cysteine and another nephrotoxic cysteine S-conjugate, S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, inactive purified cytosolic aspartate aminotransferase and purified alanine aminotransferase. These cysteine S-conjugates also inactive aspartate aminotransferase in cytosolic and mitochondrial fractions of rat brain and kidney. The present results suggest that some halogenated xenobiotics may be toxic in part through their conversion to the corresponding cysteine S-conjugate which inactivates key pyridoxal 5'-phosphate-containing enzyme.

Alanine Transaminase↗

PubMed1992

L-cysteine and S-(1,2-dichlorovinyl)-L-cysteine transport in rat liver canalicular membrane vesicles: potential reabsorption mechanisms for biliary metabolites of glutathione and its S-conjugates.

Transport of L-cysteine and a cysteine S-conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC) was investigated in rat liver canalicular plasma membrane (cLPM) vesicles. Cysteine uptake into an osmotically active intravesicular space was temperature sensitive and further enhanced by an inwardly directed Na+ gradient. Na(+)-dependent and -independent L-cysteine uptake exhibited saturation kinetics with apparent Km of 53 +/- 0.7 and 1300 +/- 300 microM and Vmax of 95 +/- 21 and 1600 +/- 200 pmol.mg protein-1.10 sec-1 for the Na(+)-dependent components, and an apparent Km of 207 +/- 48 microM and a Vmax of 355 +/- 71 pmol.mg protein-1.10 sec-1 for the Na(+)-independent component. Na(+)-dependent uptake was inhibited by L-alanine, glycine, L-phenylalanine and L-leucine, whereas Na(+)-independent uptake was inhibited by L-phenylalanine, L-leucine and 2-amino-2-norbornanecarboxylic acid. Both Na(+)-dependent and -independent L-cysteine transport processes were inhibited by several cysteine S-conjugates, with DCVC having the strongest effect. Inhibition of [35S]L-cysteine uptake by DCVC was noncompetitive with a Ki of 1.2 +/- 0.1 mM. On the other hand, uptake of [35S]DCVC by the rat cLPM vesicles was not stimulated by a Na(+)-gradient, but was inhibited by several other amino acids, including L-cysteine. Further investigation of [35S]DCVC uptake in rat cLPM vesicles indicated a saturable Na(+)-independent process with an apparent Km of 155 +/- 42 microM, and a Vmax of 393 +/- 53 pmol.mg protein-1.5 sec-1.2+.

Absorption↗

PubMed1989

Cysteine S-conjugates may act as kidney-selective prodrugs: formation of 6-mercaptopurine by the renal metabolism of S-(6-purinyl)-L-cysteine.

The recent reports that cysteine S-conjugates of several halogenated hydrocarbons are metabolized selectively in the kidney by cysteine conjugate beta-lyase (beta-lyase) to generate thiols suggest that renal beta-lyase might be exploited for site selective delivery of sulfhydryl drugs to the kidney. In this study, the in vitro and in vivo metabolism of S-(6-purinyl)-L-cysteine, the potential precursor of the antitumor and immunosuppressant drug, 6-mercaptopurine, was examined. With renal subcellular fractions, 6-mercaptopurine was identified as a metabolite of S-(6-purinyl)-L-cysteine by its electronic absorption spectrum and by high-performance liquid chromatography. The rate of enzymatic 6-mercaptopurine formation was dependent upon incubation time, substrate and protein concentrations. Aminooxyacetic acid, an inhibitor of renal beta-lyase, inhibited the reaction. The total renal beta-lyase activity with S-(6-purinyl)-L-cysteine as substrate was distributed equally between the cytosolic and mitochondrial fractions and constituted nearly 54% of the activity measured with the prototype substrate, S-(2-benzothiazolyl)-L-cysteine. The apparent Vmax value for the mitochondrial beta-lyase-dependent metabolism of S-(6-purinyl)-L-cysteine to yield 6-mercaptopurine was 4-fold greater than the rate measured with cytosol, whereas the rates of metabolism of S-(2-benzothiazolyl)-L-cysteine by the cytosolic and mitochondrial beta-lyase were similar. When rats were given S-(6-purinyl)-L-cysteine (31.2 mg/kg i.p.) and were sacrificed at 30 min after treatments, 6-mercaptopurine, 6-methyl-mercaptopurine and 6-thiouric acid were detected in the kidney, liver or plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

The role of glutathione conjugate metabolism and cysteine conjugate beta-lyase in the mechanism of S-cysteine conjugate toxicity in LLC-PK1 cells.

A cell line derived from pig kidney, LLC-PK1, was grown in a culture system in which the cells express morphological and biochemical characteristics of the proximal tubule. This model was used to investigate the mechanism of S-cysteine conjugate toxicity and the role of glutathione conjugate metabolism. LLC-PK1 cells have the degradative enzymes of the mercapturate pathway, and S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)-L-glutathione are toxic. S-(1,2-Dichlorovinyl)-L-glutathione is not toxic when the cells are pretreated with AT-125, an inhibitor of gamma-glutamyl transpeptidase. The cells respond to a variety of toxic cysteine conjugates. Cysteine conjugate beta-lyase activity is not detectable by standard assays, but can be measured using radiolabeled S-(1,2-dichlorovinyl)-L-cysteine. Pyruvate stimulates the beta-elimination reaction with S-(1,2-dichlorovinyl)-L-cysteine as substrate 2-3-fold. The data suggest that a side transamination reaction regulates the flux of substrate through the beta-elimination pathway; therefore, cysteine conjugate beta-lyase in LLC-PK1 cells may be regulated by transamination, and measurement of lyase activity in some systems may require the presence of alpha-ketoacids. Aminoxyacetic acid blocks both the metabolism of S-(1,2-dichlorovinyl)-L-cysteine to a reactive species which covalently binds to cellular macromolecules and toxicity. Glutathione inhibits the binding of the sulfur containing cleavage fragment to acid insoluble material in vitro. The data provide direct evidence that S-(1,2-dichlorovinyl)-L-cysteine is metabolized to a reactive species which covalently binds to cellular macromolecules, and the binding is proportional to toxicity.

Acetylcysteine↗

PubMedDate not supplied

Spinach threonine dehydratase. Inhibition by L-cysteine and D-cysteine.

L-threonine deaminase from spinach is inhibited by D- and L-cysteine. The inhibition patterns by D-cysteine and by L-cysteine are non-competitive. The value of Ki for D-cysteine and L-cysteine is of the same order of magnitude. Inhibitions by L-isoleucine and L-cysteine are additional. These results indicate that inhibition by L-cysteine occurs on a site of enzyme which is different from the binding site for L-isoleucine and L-valine. Probably L-cysteine and D-cysteine form the thiazolidinic ring with a PLP mole. Which is not costituent of the active site, but located in a different region.

Binding Sites↗

PubMed2005

Induction of cysteine dioxygenase activity by oral administration of cysteine analogues to the rat: implications for drug efficacy and safety.

One of the major steps in the oxidation of the sulphur-containing amino acid, L-cysteine, is the production of cysteine sulphinic acid, catalysed by the enzyme cysteine dioxygenase. This enzyme plays a key role in the intermediary metabolism of sulphur-containing compounds. The activity of this crucial enzyme is known to be influenced by sulphur-compound intake, being increased in animals fed an excess of L-cysteine or methionine. However, the affects on this enzyme of the chronic administration of drugs similar in structure to cysteine are unknown. This has now been investigated using the anti-rheumatic agent, D-penicillamine, and the mucoactive compound, S-carboxymethyl-L-cysteine. Repeated oral administration of these sulphur-containing drugs to male Wistar rats for five consecutive days led to a significant increase in hepatic cysteine dioxygenase activity. This increase in the production rate of cysteine sulphinic acid remained evident until returning to control levels four days after cessation of drug administration. These observations provide evidence that these two drugs interact with the intermediary biochemistry of sulphur compounds and may provide hitherto unappreciated insights into mechanisms by which therapeutic effects and adverse reactions may occur.

Administration, Oral↗