Novel applications of modification of thiol enzymes and redox-regulated proteins using S-methyl methanethiosulfonate (MMTS).

S-Methyl methanethiosulfonate (MMTS) is used in experimental biochemistry for alkylating thiol groups of protein cysteines. Its applications include mainly trapping of natural thiol-disulfide states of redox-sensitive proteins and proteins which have undergone S-nitrosylation. The reagent can also be employed as an inhibitor of enzymatic activity, since nucleophilic cysteine thiolates are commonly present at active sites of various enzymes. The advantage of using MMTS for this purpose is the reversibility of the formation of methylthio mixed disulfides, compared to irreversible alkylation using conventional agents. Additional benefits include good accessibility of MMTS to buried protein cysteines due to its small size and the simplicity of the protection and deprotection procedures. In this study we report examples of MMTS application in experiments involving oxidoreductase (glyceraldehyde-3-phosphate dehydrogenase, GAPDH), redox-regulated protein (recoverin) and cysteine protease (triticain-α). We demonstrate that on the one hand MMTS can modify functional cysteines in the thiol enzyme GAPDH, thereby preventing thiol oxidation and reversibly inhibiting the enzyme, while on the other hand it can protect the redox-sensitive thiol group of recoverin from oxidation and such modification produces no impact on the activity of the protein. Furthermore, using the example of the papain-like enzyme triticain-α, we report a novel application of MMTS as a protector of the primary structure of active cysteine protease during long-term purification and refolding procedures. Based on the data, we propose new lines of MMTS employment in research, pharmaceuticals and biotechnology for reversible switching off of undesirable activity and antioxidant protection of proteins with functional thiol groups.

Cruzain structures: apocruzain and cruzain bound to S-methyl thiomethanesulfonate and implications for drug design.

Chagas disease, which is caused by Trypanosoma cruzi, affects more than six million people worldwide. Cruzain is the major cysteine protease involved in the survival of this parasite. Here, the expression, purification and crystallization of this enzyme are reported. The cruzain crystals diffracted to 1.2 Šresolution, yielding two novel cruzain structures: apocruzain and cruzain bound to the reversible covalent inhibitor S-methyl thiomethanesulfonate. Mass-spectrometric experiments confirmed the presence of a methylthiol group attached to the catalytic cysteine. Comparison of these structures with previously published structures indicates the rigidity of the cruzain structure. These results provide further structural information about the enzyme and may help in new in silico studies to identify or optimize novel prototypes of cruzain inhibitors.

Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?

BACKGROUND AND PURPOSE: Posttranslational modifications of cysteine residues represent a major aspect of redox biology, and their reliable detection is key in providing mechanistic insights. The metastable character of these modifications and cell lysis-induced artifactual oxidation render current state-of-the-art protocols to rely on alkylation-based stabilization of labile cysteine derivatives before cell/tissue rupture. An untested assumption in these procedures is that for all cysteine derivatives, alkylation rates are faster than their dynamic interchange. However, when the interconversion of cysteine derivatives is not rate limiting, electrophilic labelling is under Curtin-Hammett control; hence, the final alkylated mixture may not represent the speciation that prevailed before alkylation. EXPERIMENTAL APPROACH: Buffered aqueous solutions of inorganic, organic, cysteine, GSH and GAPDH polysulfide species were used. Additional experiments in human plasma and serum revealed that monobromobimane can extract sulfide from the endogenous sulfur pool by shifting speciation equilibria, suggesting caution should be exercised when interpreting experimental results using this tool. KEY RESULTS: In the majority of cases, the speciation of alkylated polysulfide/thiol derivatives depended on the experimental conditions. Alkylation perturbed sulfur speciation in both a concentration- and time-dependent manner and strong alkylating agents cleaved polysulfur chains. Moreover, the labelling of sulfenic acids with dimedone also affected cysteine speciation, suggesting that part of the endogenous pool of products previously believed to represent sulfenic acid species may represent polysulfides. CONCLUSIONS AND IMPLICATIONS: We highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiological speciation of sulfur species. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Medication adherence and quality of life among the elderly with diabetic retinopathy / Adesão medicamentosa e qualidade de vida em idosos com retinopatia diabética / Adhesión a la medicación y calidad de vida en ancianos con retinopatía diabética

METHOD: one hundred (n=100) elderly outpatients with diabetic retinopathy taking antihypertensives and/or oral antidiabetics/insulin were interviewed. Adherence was evaluated by the adherence proportion and its association with the care taken in administrating medications and by the Morisky Scale. The National Eye Institute Visual Functioning Questionnaire (NEI VFQ-25) was used to evaluate HRQoL. RESULTS: most (58%) reported the use of 80% or more of the prescribed dose and care in utilizing the medication. The item "stopping the drug when experiencing an adverse event", from the Morisky Scale, explained 12.8% and 13.5% of the variability of adherence proportion to antihypertensives and oral antidiabetics/insulin, respectively. CONCLUSION: there was better HRQoL in the Color Vision, Driving and Social Functioning domains of the NEI VFQ-25. Individuals with lower scores on the NEI VFQ-25 and higher scores on the Morisky Scale presented greater chance to be nonadherent to the pharmacological treatment of diabetes and hypertension. .

OBJETIVO: investigar os fatores relacionados à adesão medicamentosa e sua relação com a qualidade de vida relacionada à saúde em idosos com retinopatia diabética. MÉTODO: foram entrevistados 100 idosos, em acompanhamento ambulatorial, em uso de anti-hipertensivos e/ou antidiabéticos orais/insulina. A adesão foi avaliada pela proporção de adesão e sua associação com os cuidados no uso dos medicamentos e pela Escala de Morisky. O National Eye Institute Visual Funcioning Questionnaire foi utilizado para avaliar a qualidade de vida relacionada à saúde. RESULTADOS: A maioria (58%) relatou o uso de 80% ou mais das doses prescritas e os cuidados na tomada dos medicamentos. O item "interromper o uso dos medicamentos por se sentir pior", da Escala de Morisky, explicou 12,8 e 13,5% da variabilidade da proporção de adesão aos anti-hipertensivos e aos antidiabéticos orais/insulina, respectivamente. CONCLUSÃO: observou-se melhor qualidade de vida relacionada à saúde nos domínios visão de cores, dirigir automóvel e apectos sociais do National Eye Institute Visual Funcioning Questionnaire. Indivíduos com menor pontuação na National Eye Institute Visual Funcioning Questionnaire e maiores escores na Escala de Morisky apresentaram maiores chances de serem não aderentes aos medicamentos do diabetes e da hipertensão arterial. .

OBJETIVO: investigar los factores relacionados a la adhesión a la medicación y su relación con la Calidad de Vida Relacionada a la Salud (CVRS) de ancianos con retinopatía diabética. MÉTODO: fueron entrevistados cien (n=100) pacientes ancianos de ambulatorio con retinopatía diabética que toman medicamentos antihipertensivos y/o antidiabéticos orales/insulina. La adhesión fue evaluada mediante la proporción de adhesión y su asociación con el cuidado tomado en la administración de medicamentos y mediante la Escala de Morisky. El National Eye Institute Visual Functioning Questionnaire (NEI VFQ-25) fue usado para evaluar la CVRS. RESULTADOS: la mayoría (58%) relató el uso de 80% o más de la dosis prescrita y cuidado con el uso de la medicación. El ítem "suspender la droga cuando vivencia un evento adverso", de la Escala de Morisky, explicó 12.8% y 13.5% de la variabilidad en la proporción de adhesión a los antihipertensivos y antidiabéticos orales/insulina, respectivamente. CONCUSIÓN: fue encontrada mejor CVRS en los dominios de Visión Cromática, Dirección y Funcionamiento Social del NEI VFQ-25. Individuos con puntuaciones menores en el NEI VFQ-25 y puntuaciones mayores en la Escala de Morisky revelaron mayor chance de no adhesión al tratamiento farmacológico de la diabetes y hipertensión. .

Anti-genotoxicity and anti-mutagenicity of Apis mellifera venom.

The search for substances able to inhibit and/or diminish the effects of genotoxic and mutagenic substances has been the target of several investigations performed in recent times. Hymenoptera venoms constitute a considerable source of substances with pharmacological potential. The present study aimed to evaluate the cytotoxic, genotoxic and anti-genotoxic, mutagenic and anti-mutagenic potentials of Apis mellifera venom in HepG2 cells. In this evaluation, the MTT test was applied to determine the most appropriate concentrations for the genotoxicity and mutagenicity tests. It was verified that the concentrations of 0.1, 0.05 and 0.01µg/mL were not cytotoxic, hence these concentrations were used in the experiments. For the evaluation of the genotoxic and mutagenic potential of the bee venom the comet assay and the micronucleus test were applied, respectively. The concentrations mentioned above presented both genotoxic and mutagenic potential for HepG2 cells and it was necessary to test lower concentrations of the venom (10pg/mL, 1pg/mL and 0.1pg/mL) for the anti-genotoxicity and anti-mutagenicity tests, which were performed subjecting the cells to the action of MMS (methyl methanesulfonate) in order to verify the ability of the venom to inhibit or diminish the action of this compound, which has a recognized action on the genetic material. Pre-, post-treatment and simultaneous treatment with and without incubation with the venom were performed. It was observed that the lowest three concentrations tested did not present any anti-genotoxic and anti-mutagenic activity on the cells. The use of bee venom for pharmacological purposes in treatments such as cancer must be done with extreme caution, since it was observed that even at very low concentrations the venom can induce genotoxicity and mutagenicity in human cells, as was verified for the HepG2 cells.

Thiol-blocking electrophiles interfere with labeling and detection of protein sulfenic acids.

Cellular exposure to reactive oxygen species induces rapid oxidation of DNA, proteins, lipids and other biomolecules. At the proteome level, cysteine thiol oxidation is a prominent post-translational process that is implicated in normal physiology and numerous pathologies. Methods for investigating protein oxidation include direct labeling with selective chemical probes and indirect tag-switch techniques. Common to both approaches is chemical blocking of free thiols using reactive electrophiles to prevent post-lysis oxidation or other thiol-mediated cross-reactions. These reagents are used in large excess, and their reactivity with cysteine sulfenic acid, a critical oxoform in numerous proteins, has not been investigated. Here we report the reactivity of three thiol-blocking electrophiles, iodoacetamide, N-ethylmaleimide and methyl methanethiosulfonate, with protein sulfenic acid and dimedone, the structural core of many sulfenic acid probes. We demonstrate that covalent cysteine -SOR (product) species are partially or fully susceptible to reduction by dithiothreitol, tris(2-carboxyethyl)phosphine and ascorbate, regenerating protein thiols, or, in the case of ascorbate, more highly oxidized species. The implications of this reactivity on detection methods for protein sulfenic acids and S-nitrosothiols are discussed.

Propofol binding to the resting state of the gloeobacter violaceus ligand-gated ion channel (GLIC) induces structural changes in the inter- and intrasubunit transmembrane domain (TMD) cavities.

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428-431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.

Persulfide reactivity in the detection of protein s-sulfhydration.

Hydrogen sulfide (H2S) has emerged as a new member of the gaseous transmitter family of signaling molecules and appears to play a regulatory role in the cardiovascular and nervous systems. Recent studies suggest that protein cysteine S-sulfhydration may function as a mechanism for transforming the H2S signal into a biological response. However, selective detection of S-sulfhydryl modifications is challenging since the persulfide group (RSSH) exhibits reactivity akin to other sulfur species, especially thiols. A modification of the biotin switch technique, using S-methyl methanethiosulfonate (MMTS) as an alkylating reagent, was recently used to identify a large number of proteins that may undergo S-sulfhydration, but the underlying mechanism of chemical detection was not fully explored. To address this key issue, we have developed a protein persulfide model and analogue of MMTS, S-4-bromobenzyl methanethiosulfonate (BBMTS). Using these new reagents, we investigated the chemistry in the modified biotin switch method and examined the reactivity of protein persulfides toward different electrophile/nucleophile species. Together, our data affirm the nucleophilic properties of the persulfide sulfane sulfur and afford new insights into protein S-sulfhydryl chemistry, which may be exploited in future detection strategies.

Cloning, overexpression and characterization of Leishmania donovani triosephosphate isomerase.

Triosephosphate isomerase (TIM) is a major enzyme in the glycolytic pathway, which catalyzes the interconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. Here, we report cloning, expression and purification of a catalytically active recombinant TIM of Leishmania donovani (LdTIM). The recombinant LdTIM had a pH optimum in the range of 7.2-9.0, found stable at 25°C for 30 min and K(m) and V(max) for the substrate glyceraldehyde 3-phosphate was 0.328±0.02mM and 10.05mM/min/mg, respectively. The cysteine-reactive agent methylmethane thiosulphonate (MMTS) was used as probe, in order to test its effect on enzyme activity. The MMTS induced 75% enzyme inactivation within 15 min at 250 µM concentration. The biochemical characterization of LdTIM described in this work is the essential step towards deeper understanding of its role in parasite survival. The purification of LdTIM in bioactive form provides important tools for further functional and structural studies.

The TM2 6' position of GABA(A) receptors mediates alcohol inhibition.

Ionotropic GABA(A) receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABA(A)Rs. We used Xenopus laevis oocytes to investigate the 6' position in the second transmembrane region of GABA(A)Rs as a site influencing alcohol inhibition. We asked whether modification of the 6' position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6' position in either α2 or ß2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6' position. We verified a role for the 6' position in previously tested α2ß2 as well as more physiologically relevant α2ß2γ2s GABA(A)Rs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABA(A)R homolog, revealing that the 6' position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6' positions in both α2 and ß2 GABA(A)R subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels.

Identification of multiple folding pathways of monellin using pulsed thiol labeling and mass spectrometry.

Protein folding reactions often display multiexponential kinetics of changes in intrinsic optical signals, as a manifestation of heterogeneity, either on one folding pathway or on multiple folding pathways. Delineating the origin of this heterogeneity is difficult because different coexisting structural forms of a protein cannot be easily distinguished by optical probes. In this study, the complex folding reaction of single-chain monellin has been investigated using a pulsed thiol labeling (SX) methodology in conjunction with mass spectrometry, which measures the kinetics of burial of a cysteine side chain thiol during folding. Because it can directly distinguish between unfolded and folded molecules and can measure the disappearance of the former during folding, the pulsed SX methodology is an ideal method for investigating whether multiple pathways are operative during folding. The kinetics of burial of the C42 thiol of monellin was observed to follow biexponential kinetics. To determine whether this was because the fast phase leads to the partial protection of the thiol group in all the molecules or to complete protection in only a fraction of the molecules, the duration and intensity of the labeling pulse were varied. The observation that the extent of labeling did not vary with the duration of the pulse cannot be explained by a simple sequential folding mechanism. Two parallel folding pathways are shown to be operative, with one leading to the formation of thiol-protective structure more rapidly than the other.

Chelator-facilitated chemical modification of IMP-1 metallo-beta-lactamase and its consequences on metal binding.

A method involving the reversible chemical modification of an active site, zinc-binding cysteine residue (Cys221) for the specific removal of one of the two zinc ions in the metallo-beta-lactamase IMP-1 was explored. Covalent modification of Cys221 by 5,5'-dithio-bis(2-nitrobenzoic acid) was greatly enhanced by the presence of dipicolinic acid, and subsequent removal of the modifying group was easily achieved by reduction of the disulfide bond. However, mass spectrometric analyses and an assessment of IMP-1's catalytic competence are consistent with the maintenance of the enzyme's binuclear status. The consequences arising from chemical modification of Cys221 are thus distinct from those reported for Cys-->Ala/Ser mutants of IMP-1 and other metallo-beta-lactamases, which are mononuclear.

Species-specific inhibition of Giardia lamblia triosephosphate isomerase by localized perturbation of the homodimer.

Giardia lamblia depends on glycolysis to obtain ATP, highlighting the suitability of glycolytic enzymes as targets for drug design. We studied triosephosphate isomerase from G. lamblia (GlTIM) as a potential species-specific drug target. Cysteine-reactive agents were used as probes, in order to test those regions near to cysteine residues as targets to perturb enzyme structure and activity. Methyl methanethiosulfonate (MMTS) derivatized three of the five Cys per subunit of dimeric GlTIM and induced 50% of inactivation. The 2-carboxyethyl methanethiosulfonate (MTSCE) modified four Cys and induced 97% of inactivation. Inactivation by MMTS or MTSCE did not affect secondary structure, nor induce dimer dissociation; however, Cys modification decreased thermal stability of enzyme. Inactivation and dissociation of the dimer to stable monomers were reached when four Cys were derivatized by 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). The effects of DTNB were completely abolished when GlTIM was first treated with MMTS. The effect of thiol reagents on human TIM was also assayed; it is 180-fold less sensitive than GlTIM. Collectively, the data illustrate GlTIM as a good target for drug design.

Gas-phase structure, rotational barrier, and vibrational properties of methyl methanethiosulfonate, CH3SO2SCH3: an experimental and computational study.

The molecular structure of methyl methanethiosulfonate, CH3SO2SCH3, has been determined in the gas phase from electron-diffraction data supplemented by ab initio (HF, MP2) and density functional theory (DFT) calculations using 6-31G(d), 6-311++G(d,p), and 6-311G(3df,3pd) basis sets. Both experimental and theoretical data indicate that although both anti and gauche conformers are possible by rotating about the S-S bond, the preferred conformation is gauche. The barrier to internal rotation in the CSSC skeleton has been calculated using the RHF/6-31G(d), MP2/6-31G(d), and B3LYP/6-31G(d) methods as well as MP2 with a 6-31G(3df) basis set on sulfur and 6-31G(d) on C, H, and O. A 6-fold decomposition of the rotational barrier has been performed in terms of a Fourier-type expansion, enabling us to analyze the nature of the potential function, showing that the coefficients V1 and V2 are the dominant terms; V1 is associated with nonbonding interactions, and V2 is associated with hyperconjugative interactions. A natural bond orbital analysis showed that the lone pair --> sigma* hyperconjugative interactions favor the gauche conformation. Furthermore, the infrared spectra for the liquid and solid phases and the Raman spectrum for the liquid have been recorded, and the observed bands have been assigned to the vibrational normal modes. The experimental vibrational data, along with calculated theoretical force constants, were used to define a scaled quantum mechanical force field for the target system that enabled us to estimate the measured frequencies with a final root-mean-square deviation of 6 cm-1.

Control of inward rectifier K channel activity by lipid tethering of cytoplasmic domains.

Interactions between nontransmembrane domains and the lipid membrane are proposed to modulate activity of many ion channels. In Kir channels, the so-called "slide-helix" is proposed to interact with the lipid headgroups and control channel gating. We examined this possibility directly in a cell-free system consisting of KirBac1.1 reconstituted into pure lipid vesicles. Cysteine substitution of positively charged slide-helix residues (R49C and K57C) leads to loss of channel activity that is rescued by in situ restoration of charge following modification by MTSET(+) or MTSEA(+), but not MTSES(-) or neutral MMTS. Strikingly, activity is also rescued by modification with long-chain alkyl-MTS reagents. Such reagents are expected to partition into, and hence tether the side chain to, the membrane. Systematic scanning reveals additional slide-helix residues that are activated or inhibited following alkyl-MTS modification. A pattern emerges whereby lipid tethering of the N terminus, or C terminus, of the slide-helix, respectively inhibits, or activates, channel activity. This study establishes a critical role of the slide-helix in Kir channel gating, and directly demonstrates that physical interaction of soluble domains with the membrane can control ion channel activity.

Identification of sequences that target BRCA1 to nuclear foci following alkylative DNA damage.

BRCA1 is a tumor suppressor involved in the maintenance of genome integrity. BRCA1 co-localizes with DNA repair proteins at nuclear foci in response to DNA double-strand breaks caused by ionizing radiation (IR). The response of BRCA1 to agents that elicit DNA single-strand breaks (SSB) is poorly defined. In this study, we compared chemicals that induce SSB repair and observed the most striking nuclear redistribution of BRCA1 following treatment with the alkylating agent methyl methanethiosulfonate (MMTS). In MCF-7 breast cancer cells, MMTS induced movement of endogenous BRCA1 into distinctive nuclear foci that co-stained with the SSB repair protein XRCC1, but not the DSB repair protein gamma-H2AX. XRCC1 did not accumulate in foci after ionizing radiation. Moreover, we showed by deletion mapping that different sequences target BRCA1 to nuclear foci induced by MMTS or by ionizing radiation. We identified two core MMTS-responsive sequences in BRCA1: the N-terminal BARD1-binding domain (aa1-304) and the C-terminal sequence aa1078-1312. These sequences individually are ineffective, but together they facilitated BRCA1 localization at MMTS-induced foci. Site-directed mutagenesis of two SQ/TQ motif serines (S1143A and S1280A) in the BRCA1 fusion protein reduced, but did not abolish, targeting to MMTS-inducible foci. This is the first report to describe co-localization of BRCA1 with XRCC1 at SSB repair foci. Our results indicate that BRCA1 requires BARD1 for targeting to different types of DNA lesion, and that distinct C-terminal sequences mediate selective recruitment to sites of double- or single-strand DNA damage.

Does s-methyl methanethiosulfonate trap the thiol-disulfide state of proteins?

S-Methyl methanethiosulfonate (MMTS) is a reagent used to trap the natural thiol-disulfide state of the proteins. The efficiency of trapping mixed disulfides in vivo has been found to be higher for MMTS than for the more commonly used N-ethylmaleimide. MMTS has also been used for studying protein S-nitrosylation and the role of catalytic and structural cysteines on enzyme activities. However, the treatment of a protein with MMTS can potentially generate additional protein disulfide bonds. These results indicate that in vitro MMTS is able to form both intramolecular and intermolecular protein disulfide bonds in addition to dithiomethane adducts.

Crosstalk between the subunits of the homodimeric enzyme triosephosphate isomerase.

Homodimeric triosephosphate isomerase (TIM) from Trypanosoma cruzi (TcTIM) and T. brucei (TbTIM) are markedly similar in amino acid sequence and three-dimensional structure. In their dimer interfaces, each monomer has a Cys15 that is surrounded by loop3 of the adjoining subunit. Perturbation of Cys15 by methylmethane thiosulfonate (MMTS) induces abolition of catalysis and structural changes. In the two TIMs, the structural arrangements of their Cys15 are almost identical. Nevertheless, the susceptibility of TcTIM to MMTS is nearly 100-fold higher than in TbTIM. To ascertain the extent to which the characteristics of the interface Cys depend on the dynamics of its own monomer or on those of the adjacent monomer, we studied MMTS action on mutants of TcTIM that had the interface residues of TbTIM, and hybrids that have only one interfacial Cys15 (C15ATcTIM-wild type TbTIM). We found that the solvent exposure of the interfacial Cys depends predominantly on the characteristics of the adjoining monomer. The maximal inhibition of activity induced by perturbation of the sole interface Cys in the C15ATcTIM-TbTIM hybrid is around 60%. Hybrids formed with C15ATcTIM monomers and catalytically inert TbTIM monomers (E168DTbTIM) were also studied. Their activity drops by nearly 50% when the only interfacial Cys is perturbed. These results in conjunction with those on C15ATcTIM-wild type TbTIM hybrid indicate that about half of the activity of each monomer depends on the integrity of each of the two Cys15-loop3 portions of the interface. This could be another reason of why TIM is an obligatory dimer.

Identification of a stable flavin-thiolate adduct in heterotetrameric sarcosine oxidase.

Heterotetrameric sarcosine oxidase (TSOX) is a complex bifunctional flavoenzyme that contains two flavins. Most of the FMN in recombinant TSOX is present as a covalent adduct with an endogenous ligand. Enzyme denaturation disrupts the adduct, accompanied by release of a stoichiometric amount of sulfide. Enzyme containing>or=90% unmodified FMN is prepared by displacement of the endogenous ligand with sulfite, a less tightly bound competing ligand. Reaction of adduct-depleted TSOX with sodium sulfide produces a stable complex that resembles the endogenous TSOX adduct and known 4a-S-cysteinyl flavin adducts. The results provide definitive evidence for sulfide as the endogenous TSOX ligand and strongly suggest that the modified FMN is a 4a-sulfide adduct. A comparable reaction with sodium sulfide is not detected with other flavoprotein oxidases. A model of the postulated TSOX adduct suggests that it is stabilized by nearby residues that may be important in the electron transferase/oxidase function of the coenzyme.

Disulfiram irreversibly aggregates betaine aldehyde dehydrogenase--a potential target for antimicrobial agents against Pseudomonas aeruginosa.

In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacterium against the high-osmolality stress prevalent in the infected tissues. This tetrameric enzyme contains four cysteine residues per subunit and is a potential drug target. In our search for specific inhibitors, we mutated the catalytic Cys286 to alanine and chemically modified the recombinant wild-type and the four Cys-->Ala single mutants with thiol reagents. The small methyl-methanethiosulfonate inactivated the enzymes without affecting their stability while the bulkier dithionitrobenzoic acid (DTNB) and bis[diethylthiocarbamyl] disulfide (disulfiram) induced enzyme dissociation--at 23 degrees C--and irreversible aggregation--at 37 degrees C. Of the four Cys-->Ala mutants only C286A retained its tetrameric structure after DTNB or disulfiram treatments, suggesting that steric constraints arising upon the covalent attachment of a bulky group to C286 resulted in distortion of the backbone configuration in the active site region followed by a severe decrease in enzyme stability. Since neither NAD(P)H nor betaine aldehyde prevented disulfiram-induced PaBADH inactivation or aggregation, and reduced glutathione was unable to restore the activity of the modified enzyme, we propose that disulfiram could be a useful drug to combat infection by P. aeruginosa.