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PubMed1988

Identification of S-(2,5-dihydroxyphenyl)-cysteine and S-(2,5-dihydroxyphenyl)-N-acetyl-cysteine as urinary metabolites of acetaminophen in the mouse. Evidence for p-benzoquinone as a reactive intermediate in acetaminophen metabolism.

S-(2,5-Dihydroxyphenyl)-cysteine and S-(2,5-dihydroxyphenyl)-N-acetyl-cysteine [the cysteine- and N-acetyl-cysteine adducts, respectively, of hydroquinone (HQ)] were identified and quantified in the urine of mice administered [ring-U-14C]acetaminophen [14C]APAP, 200 mg kg-1, i.p.). Urine was collected for 24 h and fractionated by HPLC to isolate the above adducts. These conjugates were then converted to a common derivative, viz. O,O',S-tris-acetyl-3-thio-hydroquinone, which was characterized by GC/MS. Neither of the HQ adducts was detected in the urine of control mice which had not received APAP. Quantification of urinary HQ-cysteine and HQ-N-acetyl-cysteine was performed by HPLC techniques, which indicated that these conjugates accounted for approx. 1.5% of the administered dose of APAP after 24 h, a figure which is equivalent to 6.3% of the corresponding APAP-thiol conjugates in the urine. These findings provide strong indirect evidence that p-benzoquinone is formed as a reactive, but apparently non-hepatotoxic, metabolite of APAP in vivo.

Acetaminophen↗

PubMed1992

Topological analysis of the hepatitis B virus core particle by cysteine-cysteine cross-linking.

The nucleocapsid, or core particle, of hepatitis B virus is formed by 180 subunits of the core protein, which contains Cys at positions 48, 61, 107 and 183, the latter constituting the C terminus. Upon adventitious oxidation, some or all of these cysteine residues participate in the formation of disulphide bridges, leading to polymerization of the subunits within the particle. To utilize the cysteine residues as topological probes, we reduced the number of possible intersubunit crosslinks by replacing these residues individually, or in all combinations, by serine. A corresponding set of variants was constructed within the context of an assembly-competent core protein variant that lacks the highly basic C-terminal region. Analysis, by polyacrylamide gel electrophoresis under non-reducing conditions, of the oxidative crosslinking products formed by the wild-type and mutant proteins expressed in Escherichia coli, revealed a clear distinction between the three N-proximal, and the C-terminal Cys: N-proximal Cys formed intermolecular disulphide bonds only with other N-proximal cysteine residues, leading to dimerization. Cys48 and Cys61, in contrast to Cys107, could be crosslinked to the homologous cysteine residues in a second subunit, and are therefore located at the dimer interface. Cys 183 predominantly formed disulphide bonds with Cys183 in subunits other than those crosslinked by the N-proximal cysteine residues. Hence, the polymers generated by oxidation of the wild-type protein are S-S-linked dimeric N-terminal domains interconnected via Cys183/Cys183 disulphide bonds. The intermolecular crosslinks between the N-proximal cysteine residues were apparently the same in the C-terminally truncated and in the full-length proteins, corroborating the model in which the N-terminal domain and the C terminus of the HBV core protein form two distinct and structurally independent entities. The strong tendency of the N-terminal domain for dimeric interactions suggests that core protein dimers are the major intermediates in hepatitis B virus nucleocapsid assembly.

Amino Acid Sequence↗

PubMed1989

N-acetyl-L-tyrosine and N-acetyl-L-cysteine as tyrosine and cysteine precursors during intravenous infusion in humans.

The usefulness of N-acetyl-L-tyrosine (NAT) and N-acetyl-L-cysteine (NAC) as tyrosine and cysteine precursors during intravenous infusion was investigated in humans. Plasma levels and urinary excretion of NAT, tyrosine, NAC, and total cysteine were determined, and the site of deacetylation was examined by measuring the splanchnic and renal balances. Eleven healthy volunteers were given 5 g of either NAT or NAC as a 4-hour intravenous infusion. Plasma levels of NAT and NAC increased rapidly, accompanied by a 25% increase in tyrosine levels and a 35% decrease in total cysteine. Urinary excretion of NAT and NAC in 4 hours accounted for 56% and 11% of the infused amount, respectively. No net production of tyrosine or cysteine was found from the splanchnic area, but from the kidneys there was a small release of both tyrosine (10 +/- 3 mumol/min) and cysteine (64 +/- 3 mumol/min). We conclude that under these conditions the usefulness of NAT and NAC as precursors for the corresponding amino acids in humans is not apparent.

Acetylcysteine↗

PubMed2005

Determination of the essentiality of the eight cysteine residues of the NrtA protein for high-affinity nitrate transport and the generation of a functional cysteine-less transporter.

All eight cysteine residues, C90, C94, C143, C147, C219, C325, C367, and C431, present in transmembrane domains of the Aspergillus nidulans NrtA nitrate transporter protein were altered individually by site-specific mutagenesis. The results indicate that six residues, C90, C147, C219, C325, C367, and C431, are not required for nitrate transport. Although alterations of C94 and C143 are less well tolerated, these residues are not mandatory and their possible role is discussed. A series of constructs, all completely devoid of cysteine residues, was generated to permit future cysteine-scanning mutagenesis. The optimum cysteine-less combination was identified as C90A, C94A, C143A, C147T, C219S, C325S, C367S, and C431S. This mutant combination yielded transformant strains with up to 40% of wild-type nitrate transport activity. Furthermore, the K(m) value and the level of protein expression were found to be similar to those of the wild-type. This cysteine-less vector should allow us to investigate in detail potentially interesting NrtA amino acids (e.g. identified from homology comparisons) which may be involved in transport, by altering these singly to cysteine and studying such residues by thiol chemistry.

Anion Transport Proteins↗

PubMed1997

Cysteine conjugate beta-lyase-dependent biotransformation of the cysteine S-conjugates of the sevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A).

2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (1, Compound A) is a fluoroalkene formed by the base-catalyzed degradation of sevoflurane that is nephrotoxic in rats. Fluoroalkene 1 is a structural analog of other nephrotoxic haloalkenes that undergo glutathione S-conjugate formation and cysteine S-conjugate beta-lyase-dependent bioactivation to reactive intermediates. The present experiments were designed to study the beta-lyase-dependent biotransformation of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (4) and S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine (5) by 19F NMR and UV spectroscopy and GC/MS. Incubation of cysteine S-conjugate 4 with rat kidney cytosol or a pyridoxal model system showed the formation of inorganic fluoride, pyruvate, and 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid (9), the expected products of a beta-lyase-catalyzed reaction. The ratio of fluoride to pyruvate ranged from 2.3 to 2.5. The amount of acid 9 formed in the rat kidney cytosol and the pyridoxal model system was, however, less than 5% of the amount of pyruvate formed. Incubation of conjugate 4 with rat kidney cytosol and analysis by 19F NMR spectroscopy showed resonances that were assigned to 3,3,3-trifluorolactic acid (10); the formation of acid 10 was observed in the pyridoxal model only after prolonged incubation (> 18 h). Lactic acid 10 was identified as a degradation product of acid 9. Cysteine S-conjugate 5 was not stable in pH 7.4 buffer and underwent a rapid cyclisation reaction (t1/2 approximately 5 min) to form 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3-thiazol e-4 -carboxylic acid (14). These data show that fluoroalkene 1-derived cysteine S-conjugates are substrates for renal beta-lyase and that acid 9 is formed as a terminal product. Acid 9 is, however, unstable and affords lactic acid 10 as a degradation product.

Anesthetics, Inhalation↗

PubMed1999

Protein footprinting at cysteines: probing ATP-modulated contacts in cysteine-substitution mutants of yeast DNA topoisomerase II.

Cysteine-substitution mutants of yeast DNA topoisomerase II were used to test footprinting of the enzyme by 2-nitro-5-thiocyanobenzoate, which cyanylates exposed cysteines in a native protein for peptide cleavage at the cyanylated sites upon unfolding and incubating the protein at pH 9. For a mutant enzyme containing a single cysteine, the extent of peptide cleavage was found to reflect the accessibility of the residue in the native protein. For proteins with multiple cysteines, however, such a correlation was obscured by the transfer of cyano groups from modified to unmodified cysteines during incubation of the unfolded protein at pH 9; accessibilities of the cysteinyl residues in a native protein could be assessed only if cyano shuffling was prevented by blocking uncyanylated sulfhydryls with a second thiol reagent. The successive use of two reagents in cysteine footprinting was applied in probing the ATP-modulated formation of contacts in yeast DNA topoisomerase II.

Amino Acid Substitution↗

PubMed1975

Mutants of Salmonella typhimurium responding to cysteine or methionine: their nature and possible role in the regulation of cysteine biosynthesis.

Nineteen mutants of Salmonella typhimurium responding to either cysteine or methionine (cym) have been identified amongst cysteine (cys) and methionine (met) auxotrophs. Their growth responses to known intermediates in the related pathways of cysteine and methionine biosynthesis and complementation patterns in abortive transduction tests divided the mutants into six groups. Results of conjugation, cotransduction and deletion mapping experiments substantiated these groups, each of which carried a lesion within known cys genes. Enzyme assays on cym mutants from five of the six groups confirmed their cys gene deficiencies. Growth response and enzyme assay data were not consistent with mutants being leaky cys mutants (spared by methionine). None of eight cym mutants tested were able to convert [35S]methionine into [35S]cysteine. Selenate specifically inhibits the early enzymes of cysteine synthesis. In cym mutants this inhibition was relieved by cysteine but not by methionine, indicating that cym mutants require active cys enzymes for growth on methionine. There was evidence that methionine stimulated in vivo activity of cys enzymes in a cym mutant. Resistance to inhibition by 1,2,4-triazole results in reduced levels of the O-acetyl serine sulphydrylase. In cym mutants triazole resistance gave unstable suppression of the cym phenotype. Cym mutants may result from mutation in regulatory regions common to each of the cys genes, with the precise role of methionine as yet unknown.

Adenine Nucleotides↗

PubMed2004

Structure-function analysis of the cysteine string protein in Drosophila: cysteine string, linker and C terminus.

Cysteine string proteins (CSPs) are conserved secretory vesicle proteins involved in regulating neurotransmitter and peptide release. While the function of the J-domain has been studied in detail, little is known about other conserved regions. We have constructed mutant genes coding for proteins with modified cysteine string, linker region or C terminus and transformed them into Csp null-mutant Drosophila: In the living animal, mutated CSP lacking all cysteines fails to associate with membranes, does not concentrate in synaptic terminals, and cannot rescue adult temperature-sensitive paralysis and short life span, both prominent null mutant phenotypes. A mutant protein with 5 instead of 11 string cysteines appears to be normally targeted but cannot rescue paralysis at 37 degrees C. We propose that the cysteine string, in addition to its role in targeting, may be essential for a function of CSP that is dependent on the number of cysteines in the string. A deletion in the linker region or the C terminus does not affect CSP targeting, and function in adults is only marginally impaired.

Amino Acid Sequence↗

PubMed2004

The effect of cysteine analogues on the excretion of urinary sulphate in the rat following cysteine administration.

A major pathway for the production of sulphate within the mammalian body is known to be via the oxidative degradation of the sulphur moiety within the amino acid, L-cysteine. The ability of two structurally similar sulphur-containing drugs, the anti-rheumatic agent, D-penicillamine, and the mucoactive compound, S-carboxymethyl-L-cysteine, to interfere with this sulphate production was investigated. Co-administration to the male rat of D-penicillamine (p.o.) and S-carboxymethyl-L-cysteine (p.o.) with [35S]-L-cysteine (i.p.) led to a significant decrease in the subsequent urinary elimination of inorganic sulphate whilst having no measurable effect on organic sulphate excretion. The co-administration of L-valine, an amino acid not containing sulphur, had no effect. It is not known where, within the complex sequence of events surrounding the degradation of cysteine to sulphate, that D-penicillamine or S-carboxymethyl-L-cysteine may interact.

Animals↗

PubMed2000

Porcine kidney microsomal cysteine S-conjugate N-acetyltransferase-catalyzed N-acetylation of haloalkene-derived cysteine S-conjugates.

N-Acetylation of xenobiotic-derived cysteine S-conjugates is a key step in the mercapturic acid pathway. The aim of this study was to investigate the N-acetylation of haloalkene-derived S-haloalkyl and S-haloalkenyl cysteine S-conjugates by porcine kidney cysteine S-conjugate N-acetyltransferase (NAcT). A radioactive assay for the quantification of NAcT activity was developed as a new method for partial purification of the enzyme, which was necessitated by the substantial loss of activity during the immunoaffinity chromatography method. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane-sulfonate, rather than N,N-bis[3-gluconamidopropyl]deoxycholamide, was used to solubilize the NAcT from porcine kidney microsomes in the revised procedure. The partially purified NAcT was free of detectable aminoacylase activity. Although low acetyl-coenzyme A hydrolase activity was observed, its effect on the assay was minimized by addition of excess acetyl-coenzyme A in the NAcT assay mixture. Attempts to separate the residual hydrolase activity from NAcT by different chromatographic procedures were either unsuccessful or lead to inactivation of NAcT. Most of the cysteine S-conjugates studied were N-acetylated by NAcT. Although the apparent K(m) values for the cysteine S-conjugates studied differed by a factor of approximately 2.5 (124-302 microM), a greater than 15-fold difference in the apparent V(max) (0.75-15.6 nmol/h) and V(max)/K(m) (0.008-0.126 x 10(-3) l h(-1)) values was observed. These data show that a range of haloalkene-derived cysteine S-conjugates serve as substrates for pig kidney NAcT. The significant differences in cytotoxicity of these conjugates may be a result of more variable deacetylation rates of the corresponding mercapturates.

Acetyl-CoA Hydrolase↗

PubMed1988

Transport-deficient mutations in the low density lipoprotein receptor. Alterations in the cysteine-rich and cysteine-poor regions of the protein block intracellular transport.

Certain individuals with familial hypercholesterolemia (FH) produce mutant forms of the low density lipoprotein (LDL) receptor that fail to move from the endoplasmic reticulum to the Golgi complex. Here, we describe the cloning and expression of one such mutant allele, FH 429. The mutation causes a substitution of a Val for a Gly at residue 544. When recreated in an expressible cDNA, this substitution gives rise to an LDL receptor that is not transported to the cell surface and is rapidly degraded. Three previously mapped transport-deficient alleles of the LDL receptor were traced to the cysteine-rich repeats of the protein, suggesting that the generation of non-disulfide-bonded (free) cysteines might cause the block in transport. The FH 429 mutation is not located in a cysteine-rich region, however. We have attempted to test the role of cysteine by expressing mutant cDNAs that encode proteins blocked in transport and predicted to contain free cysteines. The results suggest that free cysteines are not obligatory for the blocked intracellular movement of mutant LDL receptors.

Amino Acid Sequence↗

PubMed1980

[Effects of cysteine compounds on antibiotics I. Effects of L-cysteine and its derivatives on the potency of antibiotics (author's transl)].

Various derivatives of L-cysteine obtained by conversion to an -S-S- bond in the mucoprotein by means of -SH in the chemical structure are widely used as expectorants because they show mucous dissolving action. Recently, there have been reports that L-cysteine derivatives lower the potencies of various antibiotics. Various types of antibiotics and cysteine-type expectorants are often used concomitantly for the treatment of bacterial infections in respiratory tract diseases, and any decrease in the antibiotic potency presents a major therapeutic problem. We investigated the effects of four cysteine derivatives on 12 antibiotics, ampicillin (ABPC), amoxicillin (AMPC), sulbenicillin (SBPC), cefazolin (CEZ), cephalexin (CEX), cephalothin (CET), oxytetracycline (OTC), doxycycline (DOTC), minocycline (MINO), erythromycin (EM), ribostamycin (VSM) and lincomycin (LCM), widely used clinically in vitro with the minimum inhibitory concentration (MIc) obtained by the liquid dilution method as an index. L-Cysteine, acetylcysteine, ethylcysteine and mecysteine lowered the potencies of almost all of the antibiotics at high concentrations (500 mcg/ml), but at low concentrations (12.5 mcg/ml), mecysteine lowered the potencies of only three antibiotics and L-cysteine those of only four antibiotics, while acetylcysteine decreased the potencies of six and ethylcysteine those of seven antibiotics.

Anti-Bacterial Agents↗

PubMed1995

Kinetics of interactions of para-aminohippurate, probenecid, cysteine conjugates and N-acetyl cysteine conjugates with basolateral organic anion transporter in isolated rabbit proximal renal tubules.

Kinetics of the first 15 s of para-aminohippurate (PAH) uptake across the basolateral membrane of single isolated S2 segments of rabbit proximal renal tubules and the effects of probenecid and cysteine conjugates on them were determined. For PAH uptake in control tubules, Kt (the concentration of PAH at 1/2 Jmax) was about 110 microM and Jmax (maximal rate of PAH transport) was about 6.5 pmol min-1 nl-1. In tubules preloaded with alpha-ketoglutarate (alpha-KG), thereby stimulating PAH/alpha-KG countertransport, Jmax doubled with little change in Kt. Probenecid cis-inhibited PAH uptake with an apparent Ki of about 13 to 15 microM whether or not the tubules were preloaded with alpha-KG. High probenecid concentrations cis-inhibited PAH uptake by > 98%, indicating that essentially all movement of PAH across the basolateral membrane is carrier mediated. Zwitterionic nephrotoxic cysteine conjugate, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and nontoxic cysteine conjugate, S-(2-benzothiazole)-L-cysteine (BTC) cis-inhibited PAH uptake (apparent Ki: approximately 86 microM for DCVC; approximately 37 microM for BTC) at least as effectively as their negatively charged N-acetyl derivatives (NAC-DCVC and NAC-BTC) (apparent Ki: approximately 310 microM for NAC-DCVC; approximately 35 microM for NAC-BTC). The inhibition by both DCVC and NAC-DCVC was competitive in nature. NAC-DCVC also cis-inhibited net transepithelial secretion of PAH by isolated, perfused S2 segments. The presence of DCVC and NAC-DCVC, as well as PAH itself, in the bathing medium trans-stimulated the 15 s efflux of PAH across the basolateral membrane of single S2 segments with oil-filled lumens. These data indicate that these cysteine conjugates and their N-acetyl derivatives, not only interact competitively with the PAH transporter, but are transported by it.

Acetylcysteine↗

PubMed2002

Reactive intermediate formation from the 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A)-derived cysteine S-conjugate S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine in pyridoxal model systems.

2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A) is a degradation product of the anesthetic sevoflurane and undergoes cysteine conjugate beta-lyase-dependent bioactivation to nephrotoxic metabolites in rats. The present experiments were designed to identify reactive intermediates formed from S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine, a compound A-derived cysteine S-conjugate, in two pyridoxal model systems, namely Cu2+/pyridoxal and N-dodecylpyridoxal in cetyltrimethylammonium micelles. S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine was incubated in the model systems with benzyl bromide, pentafluorobenzyl bromide, aniline, and o-phenylenediamine as trapping agents. The products were purified by TLC and identified by 19F and 1H NMR spectroscopy and by GC/MS. In the absence of trapping agents, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 3,3,3-trifluorolactic acid, which have been identified previously in biotransformation studies, were formed. With the chemical models, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropanethiolate, the expected first intermediate, was not trapped with benzyl bromide. Rather, the dehydrofluorination product 2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenylthiolate was trapped with benzyl bromide to give benzyl 2-(fluoromethoxy)-3,3,3-trifluoropropanethioate, which was formed in both chemical models. When pentafluorobenzyl bromide was used as a trapping agent, GC/MS analysis showed that the expected thiolate was trapped to give pentafluorobenzyl 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl sulfide in the N-dodecylpyridoxal model. In both chemical models, 2-(fluoromethoxy)-3,3,3-trifluorothioacyl fluoride was trapped with aniline to give N-phenyl 2-(fluoromethoxyl)-3,3,3-trifluoropropanethioamide, which cyclized to give 3-phenyl-4-thiono-5-(trifluoromethyl)-1,3-oxazolane. The results demonstrate that most of the reactive intermediates and products formed by the beta-lyase-catalyzed biotransformation of compound A-derived cysteine S-conjugates are also formed in the two chemical systems studied. Some products were, however, formed in chemical systems that have not been observed in previous in vivo and in vitro studies; it is not known whether these products are formed in biological systems and whether they contribute to the observed nephrotoxicity of cysteine S-conjugates.

Anesthetics, Inhalation↗

PubMedDate not supplied

Effects of pH, temperature, and chemical structure on the stability of S-(purin-6-yl)-L-cysteine: evidence for a novel molecular rearrangement mechanism to yield N-(purin-6-yl)-L-cysteine.

The stability of S-(purin-6-yl)-L-cysteine (SPC), a kidney-selective prodrug of 6-mercaptopurine and a putative metabolite of 6-chloropurine, was investigated under various pH and temperature conditions. At room temperature, the half-life (t 1/2) of SPC at either highly acidic (pH 3.6) or basic conditions (pH 9.6) was longer than at neutral or slightly acidic or basic conditions (pH 5.7-8.75). The primary degradation product, N-(purin-6-yl)-L-cysteine (NPC), was isolated using Sephadex LH-20 chromatography and characterized by 1H NMR and FAB/MS after derivatization with 2-iodoacetic acid. These results reveal novel stability requirements and implicate the cysteinyl amino group and the purinyl N-1 nitrogen in the mechanism of SPC rearrangement to NPC. Further evidence for this hypothesis was provided by the findings that the stability of SPC in phosphate buffer (pH 7.4) at 37 degrees C was similar to that of S-(guanin-6-yl)-L-cysteine, whereas S-(purin-6-yl)-N-acetyl-L-cysteine and S-(purin-6-yl)glutathione which have their cysteine amino groups blocked were much more stable than SPC. S-(Purin-6-yl)-L-homocysteine (SPHC) was also more stable than SPC, possibly because the formation of a 6-membered ring transition state as would be expected with SPHC is kinetically less favored than the formation of a 5-membered ring transition state as would be expected with SPC. These results may explain previous in vivo metabolism results of SPC and its analogs and may contribute to a better understanding of stability of structurally related cysteine S-conjugates.

Chromatography, High Pressure Liquid↗

PubMed1998

Acylase I-catalyzed deacetylation of N-acetyl-L-cysteine and S-alkyl-N-acetyl-L-cysteines.

The aminoacylase that catalyzes the hydrolysis of N-acetyl-L-cysteine (NAC) was identified as acylase I after purification by column chromatography and electrophoretic analysis. Rat kidney cytosol was fractionated by ammonium sulfate precipitation, and the proteins were separated by ion-exchange column chromatography, gel-filtration column chromatography, and hydrophobic interaction column chromatography. Acylase activity with NAC and N-acetyl-L-methionine (NAM), a known substrate for acylase I, as substrates coeluted during all chromatographic steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protein was purified to near homogeneity and had a subunit Mr of 43 000, which is identical with the Mr of acylase I from porcine kidney and bovine liver. n-Butylmalonic acid was a slow-binding inhibitor of acylase I and inhibited the deacetylation of NAC with a Ki of 192 +/- 27 microM. These results show that acylase I catalyzes the deacetylation of NAC. The acylase I-catalyzed deacetylation of a range of S-alkyl-N-acetyl-L-cysteines, their carbon and oxygen analogues, and the selenium analogue of NAM was also studied with porcine kidney acylase I. The specific activity of the acylase I-catalyzed deacetylation of these substrates was related to their calculated molar volumes and log P values. The S-alkyl-N-acetyl-L-cysteines with short (C0-C3) and unbranched S-alkyl substituents were good acylase I substrates, whereas the S-alkyl-N-acetyl-L-cysteines with long (>C3) and branched S-alkyl substituents were poLr acylase I substrates. The carbon and oxygen analogues of S-methyl-N-acetyl-L-cysteine and the carbon analogue of S-ethyl-N-acetyl-L-cysteine were poor acylase I substrates, whereas the selenium analogue of NAM was a good acylase I substrate.

Acetylcysteine↗

PubMed1997

Cysteine sulfinate desulfinase, a NIFS-like protein of Escherichia coli with selenocysteine lyase and cysteine desulfurase activities. Gene cloning, purification, and characterization of a novel pyridoxal enzyme.

Selenocysteine lyase (EC 4.4.1.16) exclusively decomposes selenocysteine to alanine and elemental selenium, whereas cysteine desulfurase (NIFS protein) of Azotobacter vinelandii acts indiscriminately on both cysteine and selenocysteine to produce elemental sulfur and selenium respectively, and alanine. These proteins exhibit some sequence homology. The Escherichia coli genome contains three genes with sequence homology to nifS. We have cloned the gene mapped at 63.4 min in the chromosome and have expressed, purified to homogeneity, and characterized the gene product. The enzyme comprises two identical subunits with 401 amino acid residues (Mr 43,238) and contains pyridoxal 5'-phosphate as a coenzyme. The enzyme catalyzes the removal of elemental sulfur and selenium atoms from L-cysteine, L-cystine, L-selenocysteine, and L-selenocystine to produce L-alanine. Because L-cysteine sulfinic acid was desulfinated to form L-alanine as the preferred substrate, we have named this new enzyme cysteine sulfinate desulfinase. Mutant enzymes having alanine substituted for each of the four cysteinyl residues (Cys-100, Cys-176, Cys-323, and Cys-358) were all active. Cys-358 corresponds to Cys-325 of A. vinelandii NIFS, which is conserved among all NIFS-like proteins and catalytically essential (Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720), is not required for cysteine sulfinate desulfinase. Thus, the enzyme is distinct from A. vinelandii NIFS in this respect.

Amino Acid Sequence↗

PubMed2005

Expression, purification, and kinetic characterization of recombinant rat cysteine dioxygenase, a non-heme metalloenzyme necessary for regulation of cellular cysteine levels.

Cysteine dioxygenase (CDO, EC 1.13.11.20) is a non-heme mononuclear iron enzyme that oxidizes cysteine to cysteinesulfinate. CDO catalyzes the first step in the pathway of taurine synthesis from cysteine as well as the first step in the catabolism of cysteine to pyruvate and sulfate. Previous attempts to purify CDO have been associated with partial or total inactivation of CDO. In an effort to obtain highly purified and active CDO, recombinant rat CDO was heterologously expressed and purified, and its activity profile was characterized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility, and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The approximately 40.3 kDa full-length fusion protein was purified to homogeneity using a three-column scheme, the fusion tag was then removed by digestion with factor Xa, and a final column step was used to purify homogeneous approximately 23 kDa CDO. The purified CDO had high specific activity and kinetic parameters that were similar to those for non-purified rat liver homogenate, including a Vmax of approximately 1880 nmol min-1 mg-1 CDO (kcat=43 min-1) and a Km of 0.45 mM for L-cysteine. The expression and purification of CDO in a stable, highly active form has yielded significant insight into the kinetic properties of this unique thiol dioxygenase.

Animals↗