Cysteine In Solution Research Articles

Silver(I) complex formation with cysteine, penicillamine, and glutathione.

The complex formation between silver(I) and cysteine (H2Cys), penicillamine (H2Pen), and glutathione (H3Glu) in alkaline aqueous solution was examined using extended X-ray absorption fine structure (EXAFS) and (109)Ag NMR spectroscopic techniques. The complexes formed in 0.1 mol dm(-3) Ag(I) solutions with cysteine and penicillamine were investigated for ligand/Ag(I) (L/Ag) mole ratios increasing from 2.0 to 10.0. For the series of cysteine solutions (pH 10-11) a mean Ag-S bond distance of 2.45 ± 0.02 Å consistently emerged, while for penicillamine (pH 9) the average Ag-S bond distance gradually increased from 2.40 to 2.44 ± 0.02 Å. EXAFS and (109)Ag NMR spectra of a concentrated Ag(I)-cysteine solution (C(Ag(I)) = 0.8 mol dm(-3), L/Ag = 2.2) showed a mean Ag-S bond distance of 2.47 ± 0.02 Å and δ((109)Ag) 1103 ppm, consistent with prevailing, partially oligomeric AgS3 coordinated species, while for penicillamine (C(Ag(I)) = 0.5 mol dm(-3), L/Ag = 2.0) the mean Ag-S bond distance of 2.40 ± 0.02 Å and δ((109)Ag) 922 ppm indicate that mononuclear AgS2 coordinated complexes dominate. For Ag(I)-glutathione solutions (C(Ag(I)) = 0.01 mol dm(-3), pH ∼11), mononuclear AgS2 coordinated species with a mean Ag-S bond distance of 2.36 ± 0.02 Å dominate for L/Ag mole ratios from 2.0 to 10.0. The crystal structure of the silver(I)-cysteine compound (NH4)Ag2(HCys)(Cys)·H2O (1) precipitating at pH ∼10 was solved and showed a layer structure with both AgS3 and AgS3N coordination to the cysteinate ligands. A redetermination of the crystal structure of Ag(HPen)·H2O (2) confirmed the proposed digonal AgS2 coordination environment to bridging thiolate sulfur atoms in polymeric intertwining chains forming a double helix. A survey of Ag-S bond distances for crystalline Ag(I) complexes with S-donor ligands in different AgS2, AgS2(O/N), and AgS3 coordination environments was used, together with a survey of (109)Ag NMR chemical shifts, to assist assignments of the Ag(I) coordination in solution.

Aspartic Acid-Promoted Highly Selective and Sensitive Colorimetric Sensing of Cysteine in Rat Brain

Direct selective determination of cysteine in the cerebral system is of great importance because of the crucial roles of cysteine in physiological and pathological processes. In this study, we report a sensitive and selective colorimetric assay for cysteine in the rat brain with gold nanoparticles (Au-NPs) as the signal readout. Initially, Au-NPs synthesized with citrate as the stabilizer are red in color and exhibit absorption at 520 nm. The addition of an aqueous solution (20 μL) of cysteine or aspartic acid alone to a 200 μL Au-NP dispersion causes no aggregation, while the addition of an aqueous solution of cysteine into a Au-NP dispersion containing aspartic acid (1.8 mM) causes the aggregation of Au-NPs and thus results in the color change of the colloid from wine red to blue. These changes are ascribed to the ion pair interaction between aspartic acid and cysteine on the interface between Au-NPs and solution. The concentration of cysteine can be visualized with the naked eye and determined by UV-vis spectroscopy. The signal output shows a linear relationship for cysteine within the concentration range from 0.166 to 1.67 μM with a detection limit of 100 nM. The assay demonstrated here is highly selective and is free from the interference of other natural amino acids and other thiol-containing species as well as the species commonly existing in the brain such as lactate, ascorbic acid, and glucose. The basal dialysate level of cysteine in the microdialysate from the striatum of adult male Sprague-Dawley rats is determined to be around 9.6 ± 2.1 μM. The method demonstrated here is facile but reliable and durable and is envisaged to be applicable to understanding the chemical essence involved in physiological and pathological events associated with cysteine.

Electrochemical and thermodynamic study of the electroreduction of Bi(III) ions in the presence of cysteine in solutions of different water activity

The electroreduction of Bi(III) ions in 1–8moldm−3 chlorates(VII) in the presence of cysteine at dropping mercury electrode was studied using voltammetric and impedance methods. The determined kinetic and thermodynamic parameters indicated the magnitude of the catalytic effect of cysteine and allowed us to correlate these kinetic parameters with water activity. The presence of 1×10−3moldm−3 cysteine in the solution decreased the value of ΔS0 at all the examined chlorate(VII) concentrations and suggested changes in the dynamics of the process of Bi(III) ion electroreduction. The multistage process is confirmed by the not rectilinear lnkf=f(E) dependences. In this mechanism, the chemical stages of Bi–Hg2(SR)2 or Bi–Hg(SR)2 complexes formation with the participation of cysteine, which is a mediator in the formation of these complexes, and water can be distinguished.

Reduction of Mercury from Mackerel Fillet Using Combined Solution of Cysteine, EDTA, and Sodium Chloride

An acidic solution containing mercury chelating agents to eliminate mercury in raw fish (mackerel) fillet was developed. The solution contained hydrochloric acid, sodium hydroxide, cysteine, EDTA, and NaCl. The optimum conditions for mercury reduction were achieved using response surface methodology (RSM) at cysteine concentration of 1.25%, EDTA of 275 mg/L, NaCl of 0.5%, pH of 3.75, and exposure time of 18 min. The optimized conditions produced a solution which can remove up to 91% mercury from raw fish fillet. Cysteine and EDTA were identified as potential chelating agents with the greatest potential for use. The solution can be employed in fish industries to reduce mercury in highly contaminated fish.

Quartz Crystal Microbalance Aptasensor for Sensitive Detection of Mercury(II) Based on Signal Amplification with Gold Nanoparticles

We show that a short mercury-specific aptamer (MSA) along with gold nanoparticles (Au-NPs) can be used to determine Hg(II) ion by a combination of a QCM-based sensor and a flow system. The MSA binds specifically to Hg(II), and the Au-NPs can amplify the signal to enhance sensitivity. Specifically, the short thiolated MSAs are immobilized on the surface of the QCM as the capture probe, and the MSAs are linked to the Au-NPs as the linking probe. The two components can form a sandwich structure of the T-Hg(II)-T type in the presence of Hg(II) ions. This leads to change in the mass on the QCM and a change in the resonance frequency. Hg(II) can be determined with a detection limit of 0.24 ± 0.06 nM which is better by three orders of magnitude than previous methods. The sensor can be regenerated by disrupting the T-Hg(II)-T base pairs with a solution of cysteine.

Electrical properties of polycrystalline GaN films functionalized with cysteine and stabilization of GaN nanoparticles in aqueous media

GaN was synthesized onto sapphire substrates by chemical vapor deposition, reacting gallium, ammonium chloride and ammonia. The polycrystalline films were immersed in glycine, aspartic acid and cysteine solutions. Cysteine chemisorbed onto GaN films produced detectable changes in conductivity, mobility and Hall coefficient indicating that GaN is capable of detecting and reacting with thiolate groups, which was confirmed by X-ray photoelectron spectroscopy. The Cys-GaN film solution was adjusted to pH 10, upon which the GaN nanoparticles were transferred to the aqueous phase forming a suspension stable for seven days. The alkaline colloid was then further adjusted down to pH 3 retaining stability for three days. The GaN colloid obtained represents a suitable medium to study GaN properties for biological applications.

Investigation of reactions postulated to occur during inhibition of ribonucleotide reductases by 2′-azido-2′-deoxynucleotides

Model 3′-azido-3′-deoxynucleosides with thiol or vicinal dithiol substituents at C2′ or C5′ were synthesized to study reactions postulated to occur during inhibition of ribonucleotide reductases by 2′-azido-2′-deoxynucleotides. Esterification of 5′-(tert-butyldiphenylsilyl)-3′-azido-3′-deoxyadenosine and 3′-azido-3′-deoxythymidine (AZT) with 2,3-S-isopropylidene-2,3-dimercaptopropanoic acid or N-Boc-S-trityl-L-cysteine and deprotection gave 3′-azido-3′-deoxy-2′-O-(2,3-dimercaptopropanoyl or cysteinyl)adenosine and the 3′-azido-3′-deoxy-5′-O-(2,3-dimercaptopropanoyl or cysteinyl)thymidine analogs. Density functional calculations predicted that intramolecular reactions between generated thiyl radicals and an azido group on such model compounds would be exothermic by 33.6–41.2 kcal/mol and have low energy barriers of 10.4–13.5 kcal/mol. Reduction of the azido group occurred to give 3′-amino-3′-deoxythymidine, which was postulated to occur with thiyl radicals generated by treatment of 3′-azido-3′-deoxy-5′-O-(2,3-dimercaptopropanoyl)thymidine with 2,2′-azobis-(2-methyl-2-propionamidine) dihydrochloride. Gamma radiolysis of N2O-saturated aqueous solutions of AZT and cysteine produced 3′-amino-3′-deoxythymidine and thymine most likely by both radical and ionic processes.

An experimental study of Au removal from solution by non-metabolizing bacterial cells and their exudates

In this study, we examine the initial interactions between aqueous Au(III)-hydroxide-chloride aqueous complexes and bacteria by measuring the effects of non-metabolizing cells on the speciation and distribution of Au. We conducted batch Au(III) removal experiments, measuring the kinetics and pH dependence of Au removal, and tracking valence state transformations and binding environments using XANES spectroscopy. These experiments were conducted using non-metabolizing cells of Bacillus subtilis or Pseudomonas putida suspended in a 5ppm Au(III)-(hydroxide)-chloride starting solution of 0.1M NaClO4 to buffer ionic strength. Both bacterial species removed greater than 85% of the Au from solution after 2h of exposure time below approximately pH 5. Above pH 5, the extent of Au removed from solution decreased with increasing pH, with less than approximately 10% removal of Au from solution above pH 7.5. Kinetics experiments indicated that the Au removal with both bacterial species was rapid at pH 3, and slowed with increasing pH. Reversibility experiments demonstrated that (1) once the Au was removed from solution, adjusting 35 the pH alone did not remobilize the Au into solution and (2) the presence of cysteine in solution in the reversibility experiments caused Au to desorb, suggesting that the Au was not internalized within the bacterial cells. Our results suggest that Au removal occurs as a two-step pH-dependent adsorption reduction process. The speciation of the aqueous Au and the bacterial surface appears to control the rate of Au removal from solution. Under low pH conditions, the cell walls are only weakly negatively charged and aqueous Au complexes adsorb readily and rapidly. With increasing pH, the cell wall becomes more negatively charged, slowing adsorption significantly. The XANES data demonstrate that the reduction of Au(III) by bacterial exudates is slower and less extensive than the reduction observed in the bacteria-bearing systems, and we conclude that Au reduction occurs most rapidly and extensively upon interaction with cell wall functional groups.

Effect of nonelectrolytes on the gel formation process in aqueous silver cysteine solutions

Electron microscopy and dynamic light scattering are used to investigate the structuring process of a supramolecular system based on the aqueous solutions of L-cysteine and silver nitrate diluted with liquid water-soluble nonelectrolytes. The gel formation mechanism is discussed.

Thiol–Quinone Adduct Formation in Myofibrillar Proteins Detected by LC-MS

Protein oxidation in meat is considered to decrease meat tenderness due to protein disulfide cross-link formation of thiol-containing amino acid residues. An LC-MS method for detection of thiol-quinone adducts (RS-QH(2)) in myofibrillar proteins was developed to investigate the interaction between phenols, as protective antioxidants, and proteins from meat under oxidative conditions using aqueous solutions of (i) cysteine (Cys), (ii) glutathione (GSH), (iii) bovine serum albumin (BSA), or (iv) a myofibrillar protein isolate (MPI). The aqueous solutions were incubated at room temperature (30 min) with 4-methyl-1,2-benzoquinone (4MBQ) prepared from oxidation of 4-methylcatechol (4MC) by periodate resin or incubated at room temperature (5 h) with 4MC and Fe(II)/H(2)O(2). GSH, BSA, and MPI were hydrolyzed (6 N HCl, 110 °C, 22 h) after incubation, and the cysteine-quinone adduct, Cys-QH(2) (m/z 244.2) was identified according to UV and mass spectra after separation on an RP-C18 column. The thiol-quinone adduct was present in all thiol systems after incubation with 4MBQ or 4MC oxidized by Fe(II)/H(2)O(2). Direct reaction with 4MBQ resulted in each case in increased Cys-QH(2) formation compared to simultaneous oxidation of thiol source and 4MC with Fe(II)/H(2)O(2). The covalent bonds between quinones and thiol groups may act as a potential antioxidant by inhibiting disulfide protein cross-link formation.

Labeling, stability and biodistribution studies of 99mTc-cyclized Tyr3-octreotate derivatives

To probe the interplay between radiotracer stability and somatostatin receptor affinity, Tyr(3)-octreotate and six variations of its peptide sequence, for which the Re-cyclized products were previously reported, were radiolabeled with (99m)Tc and investigated for their in vitro stability. Radiolabeling of the peptides was effected by ligand exchange from (99m)Tc-glucoheptonate, and the desired products were purified by radio-RP-HPLC. The in vitro stability in phosphate buffered saline, mouse serum and cysteine solutions at physiological temperature and pH for all seven (99m)Tc-cyclized peptides was determined by radio-RP-HPLC and radio-TLC. Normal CF-1 mouse biodistribution studies were performed for three of the (99m)Tc-cyclized peptides. Based on the fully characterized Re-cyclized peptide analogues, four (99m)Tc-coordination motifs were proposed for the (99m)Tc-cyclized peptides. Technetium-99m-cyclized Tyr(3)-octreotate derivatives with N(2)S(2) metal coordination modes and large metal ring sizes were susceptible to oxidation and loss of (99m)Tc in the form of (99m)TcO(4)(-), as evidenced by their instability in the various solutions under physiological conditions (15-58% intact at 24 h). As anticipated, the addition of a third cysteine to the sequence stabilized the (99m)Tc metal coordination, and peptides with NS(3) coordination modes remained >85% intact out to 24 h. No significant differences were observed in the biodistribution studies performed with three peptides of varying stabilities. Improvements in stability were not sufficient to outweigh the low somatostatin receptor affinity for the peptides in this study. Further improvements in the peptide sequence and/or metal coordination are needed to result in a radiodiagnostic/radiotherapeutic pair for targeting the somatostatin receptor.

Preparation of copper and zinc sulfide nanoparticles and their modification with cysteine

A new method for the preparation of copper and zinc sulfides nanoparticles in homogeneous aqueous solutions using cysteine as a surface modifier was proposed. The size of the particles obtained is 5–7 and 1.5–3 nm for copper and zinc sulfides, respectively, depending on the concentration of the reactants. Associates of the nanoparticles 10–30 nm in size are formed in the system with an increase in the total concentrations of the sulfides. Sols of the nanoparticles obtained in cysteine solutions are resistant to oxidation and coagulation within several weeks. The variation of the synthesis conditions makes it possible to obtain zinc sulfide particles with optical properties related to size effects.

Use of polarization-dependent SERS from scratched gold films to monitor the electrochemically-driven desorption and readsorption of cysteine

The electrochemically-driven desorption and readsorption of cysteine, on scratched gold surfaces placed in a highly concentrated cysteine solution, was investigated. The ordinary Raman signal from cysteine in solution was significant relative to the SERS signal from adsorbed cysteine. However, the SERS signal was isolated from the ordinary Raman background by subtracting the signal obtained with the incident laser light polarized along the scratch (ordinary Raman) from the signal obtained with it polarized across the scratch (ordinary Raman + SERS). The method described here is applicable to other electrochemical systems where signal from solution-phase species can interfere with, or even overwhelm, signal from the adsorbate. For example, it may be used in SERS studies of water or ionic liquid structure in the double-layer region.

Detection of aminothiols through surface‐assisted laser desorption/ionization mass spectrometry using mixed gold nanoparticles

We have employed mixtures of two differently sized (average diameters: 3.5 and 14 nm) gold nanoparticles (Au NPs) as selective probes and matrices for the determination of aminothiols using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). When using 38 and 150 pM solutions of the 3.5- and 14-nm Au NPs, respectively, as the probe and matrix, SALDI-MS provided limits of detection (signal-to-noise ratio = 3) of 2, 20, and 44 nM for 1.0 mL solutions of glutathione (GSH), cysteine (Cys), and homocysteine, respectively. The signal intensities of these analytes varied by less than 20% for SALDI-MS analyses recorded over 50 sample spots; in contrast, they varied by as much as 60% when using a conventional matrix (2,5-dihydroxybenzoic acid). We validated the practicality of this approach - with its advantages of sensitivity, reproducibility, rapidity, and simplicity - through the analysis of GSH in MCF-7 cell lysates and Cys in plasma.

Biokinetic and dosimetric studies of 188Re-hyaluronic acid: a new radiopharmaceutical for treatment of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and has very limited therapeutic options. Recently, it has been found that hyaluronic acid (HA) shows selective binding to CD44 receptors expressed in most cancer histotypes. Since the trend in cancer treatment is the use of targeted radionuclide therapy, the aim of this research was to label HA with rhenium-188 and to evaluate its potential use as a hepatocarcinoma therapeutic radiopharmaceutical. (188)Re-HA was prepared by a direct labelling method to produce a ReO(O-COO)(2)-type coordination complex. (188)Re-HA protein binding and its stability in saline, phosphate buffer, human serum and cysteine solutions were determined. Biokinetic and dosimetric data were estimated in healthy mice (n=60) using the Medical Internal Radiation Dose methodology and mouse model beta-absorbed fractions. To evaluate liver toxicity, alanine aminotranferase (AST) and aspartate aminotranferase (ALT) levels in mice were assessed and the liver maximum tolerated dose (MTD) of (188)Re-HA was determined. A stable complex of (188)Re-HA was obtained with high radiochemical purity (>90%) and low serum protein binding (2%). Biokinetic studies showed a rapid blood clearance (T(1/2)alpha=21 min). Four hours after administration, (188)Re-HA was almost totally removed from the blood by the liver due to the selective uptake via HA-specific receptors (73.47+/-5.11% of the injected dose). The liver MTD in mice was approximately 40 Gy after 7.4 MBq of (188)Re-HA injection. (188)Re-HA complex showed good stability, pharmacokinetic and dosimetric characteristics that confirm its potential as a new agent for HCC radiation therapy.

Design, preparation, in vitro and in vivo evaluation of 99mTc-N 2S 2-Tat(49–57)-bombesin: A target-specific hybrid radiopharmaceutical

The gastrin-releasing peptide receptor (GRP-r) is over-expressed in various human tumors. Recently, 99mTc-EDDA/HYNIC-Lys 3-bombesin ( 99mTc-BN) was reported as a radiopharmaceutical with specific cell GRP-r binding and images in breast cancer patients demonstrated distinct radioactivity accumulation in malignant tissue. The HIV Tat-derived peptide has been used to deliver a large variety of cargoes into cells. Therefore, a new hybrid radiopharmaceutical of type 99mTc-N 2S 2-Tat(49–57)-Lys 3-bombesin ( 99mTc-Tat-BN) would increase cell uptake. The aim of this research was to prepare and assess in vitro and in vivo uptake kinetics in cancer cells of 99mTc-Tat-BN and to compare its cellular internalization with that of 99mTc-BN. Structures of N 2S 2-Tat-BN and Tc(O)N 2S 2-Tat-BN were calculated by an MM procedure. 99mTc-Tat-BN was synthesized and stability studies carried out by HPLC and ITLC-SG analyses in serum and cysteine solutions. In vitro internalization was tested using human prostate cancer PC-3 cells and breast carcinoma cell lines MDA-MB231 and MCF7. Biodistribution was determined in PC-3 tumor-bearing nude mice. Results showed a minimum energy of 271 kcal/mol for N 2S 2-Tat-BN and 300 kcal/mol for Tc(O)N 2S 2-Tat-BN. 99mTc-Tat-BN radiochemical purity was >90%. In vitro studies demonstrated stability in serum and cysteine solutions, specific cell receptor binding and internalization in three cell lines was significantly higher than that of 99mTc-BN ( p < 0.05). The tumor-to-muscle radioactivity ratio was 8.5 for 99mTc-Tat-BN and 7 for 99mTc-BN. Therefore, this hybrid is potentially useful in breast and prostate cancer imaging.

Self-assembly characteristics of gold nanoparticles in the presence of cysteine

Gold nanoparticles in aqueous dispersions were prepared by two methods, using sodium citrate as reduction agent, and their interaction with l-cysteine was investigated. UV–vis, dynamic light scattering (DLS) and zeta potential measurements were used for physical and chemical characterization of mixtures with different gold:cysteine ratios, coupled with transmission electron microscopy (TEM) and atomic force microscopy (AFM) observations. Even a diluted (0.001 M) cysteine solution leads to a rapid assembly of gold nanoparticles and a broad absorption band at longer wavelength (characteristic for the aggregation of gold nanoparticles) develops and surpasses rapidly the peak of non-aggregated gold particles. TEM images for gold nanoparticles mixed with a 0.001 M cysteine solution show randomly arranged aggregates of gold nanoparticles on TEM carbon coated copper grids. AFM images indicate a rather ordered surface of self-assembled monolayers deposited on silanized glass. From thermodynamic considerations on the protolytic equilibria in aqueous cysteine solutions, the species present at different pH values were ascertained. A possible model for cysteine binding to gold nanoparticles and for the formation of gold particle assemblies is also suggested.

Green nanotechnology from tea: phytochemicals in tea as building blocks for production of biocompatible gold nanoparticles

Phytochemicals occluded in tea have been extensively used as dietary supplements and as natural pharmaceuticals in the treatment of various diseases including human cancer. Results on the reduction capabilities of phytochemicals present in tea to reduce gold salts to the corresponding gold nanoparticles are presented in this paper. The phytochemicals present in tea serve the dual roles as effective reducing agents to reduce gold and also as stabilizers to provide robust coating on the gold nanoparticles in a single step. The Tea-generated gold nanoparticles (T-AuNPs), have demonstrated remarkable in vitro stability in various buffers including saline, histidine, HSA, and cysteine solutions. T-AuNPs with phytochemical coatings have shown significant affinity toward prostate (PC-3) and breast (MCF-7) cancer cells. Results on the cellular internalization of T-AuNPs through endocytosis into the PC-3 and MCF-7 cells are presented. The generation of T-AuNPs follows all principles of green chemistry and have been found to be non toxic as assessed through MTT assays. No 'man made' chemicals, other than gold salts, are used in this true biogenic green nanotechnological process thus paving excellent opportunities for their applications in molecular imaging and therapy.

Soybeans as a Phytochemical Reservoir for the Production and Stabilization of Biocompatible Gold Nanoparticles

The present study demonstrates an unprecedented green process for the production of gold nanoparticles by simple treatment of gold salts with soybean extracts. Reduction capabilities of antioxidant phytochemicals present in soybean and their ability to reduce gold salts chemically to nanoparticles with subsequent coating of proteins and a host of other phytochemicals present in soybean on the freshly generated gold nanoparticles are discussed. The new genre of green nanoparticles exhibit remarkable in vitro stability in various buffers including saline, histidine, HSA, and cysteine solutions. MTT assays reveal that the green gold nanoparticles are nontoxic and thus provide excellent opportunities for their applications in nanomedicine for molecular imaging and therapy. The overall strategy described herein for the generation of gold nanoparticles meets all 12 principles of green chemistry, as no "man-made" chemicals, other than the gold salts, are used in the green nanotechnological process.

Redundant Synthesis of Cysteinyl-tRNACys in Methanosarcina mazei

A subset of methanogenic archaea synthesize the cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) needed for protein synthesis using both a canonical cysteinyl-tRNA synthetase (CysRS) as well as a set of two enzymes that operate via a separate indirect pathway. In the indirect route, phosphoseryl-tRNA(Cys) (Sep-tRNA(Cys)) is first synthesized by phosphoseryl-tRNA synthetase (SepRS), and this misacylated intermediate is then converted to Cys-tRNA(Cys) by Sep-tRNA:Cys-tRNA synthase (SepCysS) via a pyridoxal phosphate-dependent mechanism. Here, we explore the function of all three enzymes in the mesophilic methanogen Methanosarcina mazei. The genome of M. mazei also features three distinct tRNA(Cys) isoacceptors, further indicating the unusual and complex nature of Cys-tRNA(Cys) synthesis in this organism. Comparative aminoacylation kinetics by M. mazei CysRS and SepRS reveals that each enzyme prefers a distinct tRNA(Cys) isoacceptor or pair of isoacceptors. Recognition determinants distinguishing the tRNAs are shown to reside in the globular core of the molecule. Both enzymes also require the S-adenosylmethione-dependent formation of (m1)G37 in the anticodon loop for efficient aminoacylation. We further report a new, highly sensitive assay to measure the activity of SepCysS under anaerobic conditions. With this approach, we demonstrate that SepCysS functions as a multiple-turnover catalyst with kinetic behavior similar to bacterial selenocysteine synthase and the archaeal/eukaryotic SepSecS enzyme. Together, these data suggest that both metabolic routes and all three tRNA(Cys) species in M. mazei play important roles in the cellular physiology of the organism.