RESUMEN
d-Penicillamine (d-Pen) is a sulfur compound used in the management of rheumatoid arthritis, Wilson's disease (WD), and alcohol dependence. Many side effects are associated with its use, particularly after long-term treatment. However, the molecular basis for such side effects is poorly understood. Based on the well-known oxidase activity of hemoproteins and the participation of catalase in cellular H2O2 redox signaling, we posit that d-Pen could inactivate catalase, thus disturbing H2O2 levels. Herein, we report on the molecular basis that could partly explain the side effects associated with this drug compound, and we demonstrate that it induces the formation of compound II, a temporarily inactive state of the enzyme, through two distinct mechanisms. Initially, d-Pen reacts with native catalase and/or iron metal ions, used to mimic non-heme iron overload observed in long-term treated WD patients, to generate thiyl radicals. These radicals partake in a futile redox cycle, thus producing superoxide radical anions O2â¢- and hydrogen peroxide H2O2. Then, either H2O2 unexpectedly reacts with reduced CAT-Fe(II) to produce compound II or both aforementioned reactive oxygen species intervene in compound II generation through compound I formation and then reduction. These findings support the evidence that d-Pen could perturb H2O2 redox homeostasis through transient but recurring catalase inactivation, which may in part rationalize some deleterious effects observed with this therapeutic agent, as discussed.
Asunto(s)
Peróxido de Hidrógeno , Penicilamina , Catalasa , Humanos , Penicilamina/farmacología , Racionalización , SuperóxidosRESUMEN
Guanine nucleotide exchange factors (GEFs) of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies activate small GTPases of the Arf family in endocytic events. These ArfGEFs carry a pleckstrin homology (PH) domain in tandem with their catalytic Sec7 domain, which is autoinhibitory and supports a positive feedback loop in cytohesins but not in BRAGs, and has an as-yet unknown role in EFA6 regulation. In this study, we analyzed how EFA6A is regulated by its PH and C terminus (Ct) domains by reconstituting its GDP/GTP exchange activity on membranes. We found that EFA6 has a previously unappreciated high efficiency toward Arf1 on membranes and that, similar to BRAGs, its PH domain is not autoinhibitory and strongly potentiates nucleotide exchange on anionic liposomes. However, in striking contrast to both cytohesins and BRAGs, EFA6 is regulated by a negative feedback loop, which is mediated by an allosteric interaction of Arf6-GTP with the PH-Ct domain of EFA6 and monitors the activation of Arf1 and Arf6 differentially. These observations reveal that EFA6, BRAG, and cytohesins have unanticipated commonalities associated with divergent regulatory regimes. An important implication is that EFA6 and cytohesins may combine in a mixed negative-positive feedback loop. By allowing EFA6 to sustain a pool of dormant Arf6-GTP, such a circuit would fulfill the absolute requirement of cytohesins for activation by Arf-GTP before amplification of their GEF activity by their positive feedback loop.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factor 1 de Ribosilacion-ADP/química , Factor 1 de Ribosilacion-ADP/genética , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/química , Factores de Ribosilacion-ADP/genética , Sustitución de Aminoácidos , Animales , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Cricetinae , Activación Enzimática , Retroalimentación Fisiológica , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Liposomas , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfotransferasas/química , Sulfotransferasas/genética , Sulfotransferasas/metabolismoRESUMEN
Persulfides are an emerging class of cysteine oxidative post-translational modification. They react with the bioconjugation reagents bicyclo[6.1.0]nonynes (BCNs) to engender thioethers and/or disulfides. This new reactivity of BCNs with a biologically important redox-signaling species efficiently interferes with the recent usage of strained cycloalkynes to specifically trap protein sulfenic acids.
Asunto(s)
Compuestos Bicíclicos con Puentes/química , Cisteína/química , Proteínas/química , Sulfuros/química , Alquinos/química , Secuencia de Aminoácidos , Animales , Bovinos , Disulfuros/química , Datos de Secuencia Molecular , Albúmina Sérica Bovina/química , Ácidos Sulfénicos/químicaRESUMEN
Fidelity during cofactor assembly is essential for the proper functioning of metalloenzymes and is ensured by specific chaperones. MeaB, a G-protein chaperone for the coenzyme B12-dependent radical enzyme methylmalonyl-CoA mutase (MCM), uses the energy of GTP binding, hydrolysis or both to regulate cofactor loading into MCM, protect MCM from inactivation and rescue MCM that is inactivated during turnover. Typically, G proteins signal to client proteins using the conformationally mobile switch I and II loops. Crystallographic snapshots of MeaB reported herein reveal a new switch III element that has substantial conformational plasticity. Using alanine-scanning mutagenesis, we demonstrate that the switch III motif is critical for bidirectional signal transmission of the GTPase-activating protein activity of MCM and the chaperone functions of MeaB in the MeaB-MCM complex. Mutations in the switch III loop identified in patients corrupt this interprotein communication and lead to methylmalonic aciduria, an inborn error of metabolism.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Metilmalonil-CoA Mutasa/metabolismo , Chaperonas Moleculares/metabolismo , Transducción de Señal , Vitamina B 12/metabolismo , Secuencias de Aminoácidos , Humanos , Metilmalonil-CoA Mutasa/químicaRESUMEN
Hydrogen sulfide (H2S) is a gaseous signaling molecule that participates in various signaling functions in health and diseases. The tetrameric cystathionine γ-lyase (CSE) contributes to H2S biogenesis and several investigations provide evidence on the pharmacological modulation of CSE as a potential target for the treatment of a multitude of conditions. D-penicillamine (D-pen) has recently been reported to selectively impede CSE-catalyzed H2S production but the molecular bases for such inhibitory effect have not been investigated. In this study, we report that D-pen follows a mixed-inhibition mechanism to inhibit both cystathionine (CST) cleavage and H2S biogenesis by human CSE. To decipher the molecular mechanisms underlying such a mixed inhibition, we performed docking and molecular dynamics (MD) simulations. Interestingly, MD analysis of CST binding reveals a likely active site configuration prior to gem-diamine intermediate formation, particularly H-bond formation between the amino group of the substrate and the O3' of PLP. Similar analyses realized with both CST and D-pen identified three potent interfacial ligand-binding sites for D-pen and offered a rational for D-pen effect. Thus, inhibitor binding not only induces the creation of an entirely new interacting network at the vicinity of the interface between enzyme subunits, but it also exerts long range effects by propagating to the active site. Overall, our study paves the way for the design of new allosteric interfacial inhibitory compounds that will specifically modulate H2S biogenesis by cystathionine γ-lyase.
Asunto(s)
Sulfuro de Hidrógeno , Humanos , Sulfuro de Hidrógeno/metabolismo , Cistationina gamma-Liasa/metabolismo , Transducción de Señal , Cistationina , Simulación por ComputadorRESUMEN
The mechanism by which docking fidelity is achieved for the multitude of cofactor-dependent enzymes is poorly understood. In this study, we demonstrate that delivery of coenzyme B(12) or 5'-deoxyadenosylcobalamin by adenosyltransferase to methylmalonyl-CoA mutase is gated by a small G protein, MeaB. While the GTP-binding energy is needed for the editing function; that is, to discriminate between active and inactive cofactor forms, the chemical energy of GTP hydrolysis is required for gating cofactor transfer. The G protein chaperone also exerts its editing function during turnover by using the binding energy of GTP to elicit release of inactive cofactor that is occasionally formed during the catalytic cycle of MCM. The physiological relevance of this mechanism is demonstrated by a patient mutation in methylmalonyl-CoA mutase that does not impair the activity of this enzyme per se but corrupts both the fidelity of the cofactor-loading process and the ejection of inactive cofactor that forms occasionally during catalysis. Consequently, cofactor in the incorrect oxidation state gains access to the mutase active site and is not released if generated during catalysis, leading, respectively, to assembly and accumulation of inactive enzyme and resulting in methylmalonic aciduria.
Asunto(s)
Cobamidas/metabolismo , Proteínas de Unión al GTP/metabolismo , Errores Innatos del Metabolismo/orina , Ácido Metilmalónico/orina , Secuencia de Bases , Calorimetría , Cartilla de ADN , Espectroscopía de Resonancia por Spin del Electrón , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Metilmalonil-CoA Mutasa/metabolismo , Modelos Moleculares , Mutación , TermodinámicaRESUMEN
DJ-1 (also called PARK7) is a ubiquitously expressed protein involved in the etiology of Parkinson disease and cancers. At least one of its three cysteine residues is functionally essential, and its oxidation state determines the specific function of the enzyme. DJ-1 was recently reported to be persulfidated in mammalian cell lines, but the implications of this post-translational modification have not yet been analyzed. Here, we report that recombinant DJ-1 is reversibly persulfidated at cysteine 106 by reaction with various sulfane donors and subsequently inhibited. Strikingly, this reaction is orders of magnitude faster than C106 oxidation by H2O2, and persulfidated DJ-1 behaves differently than sulfinylated DJ-1. Both these PTMs most likely play a dedicated role in DJ-1 signaling or protective pathways.
Asunto(s)
Peróxido de Hidrógeno , Enfermedad de Parkinson , Animales , Humanos , Cisteína/metabolismo , Peróxido de Hidrógeno/farmacología , Mamíferos/metabolismo , Proteínas Oncogénicas/metabolismo , Oxidación-Reducción , Enfermedad de Parkinson/metabolismo , Proteína Desglicasa DJ-1/metabolismoRESUMEN
Coenzyme B(12) is used by two highly similar radical enzymes, which catalyze carbon skeleton rearrangements, methylmalonyl-CoA mutase and isobutyryl-CoA mutase (ICM). ICM catalyzes the reversible interconversion of isobutyryl-CoA and n-butyryl-CoA and exists as a heterotetramer. In this study, we have identified >70 bacterial proteins, which represent fusions between the subunits of ICM and a P-loop GTPase and are currently misannotated as methylmalonyl-CoA mutases. We designate this fusion protein as IcmF (isobutyryl-CoA mutase fused). All IcmFs are composed of the following three domains: the N-terminal 5'-deoxyadenosylcobalamin binding region that is homologous to the small subunit of ICM (IcmB), a middle P-loop GTPase domain, and a C-terminal part that is homologous to the large subunit of ICM (IcmA). The P-loop GTPase domain has very high sequence similarity to the Methylobacterium extorquens MeaB, which is a chaperone for methylmalonyl-CoA mutase. We have demonstrated that IcmF is an active ICM by cloning, expressing, and purifying the IcmFs from Geobacillus kaustophilus, Nocardia farcinica, and Burkholderia xenovorans. This finding expands the known distribution of ICM activity well beyond the genus Streptomyces, where it is involved in polyketides biosynthesis, and suggests a role for this enzyme in novel bacterial pathways for amino acid degradation, myxalamid biosynthesis, and acetyl-CoA assimilation.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Proteínas de Unión al GTP/metabolismo , Isomerasas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Vitamina B 12/metabolismo , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cobamidas/metabolismo , Biología Computacional , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/química , Regulación Bacteriana de la Expresión Génica , Isomerasas/genética , Cinética , Nucleótidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Streptomyces/enzimologíaRESUMEN
The reactivity and relative rarity of most cofactors pose challenges for their delivery to target enzymes. Using kinetic analyses, we demonstrate that adenosyltransferase, which catalyzes the final step in the assimilation of coenzyme B12, directly transfers the cofactor to methylmalonyl coenzyme A mutase. The strategy of using the final enzyme in an assimilation pathway for tailoring a cofactor and delivering it to a dependent enzyme may be general for cofactor trafficking.
Asunto(s)
Cobamidas/metabolismo , Metionina Adenosiltransferasa/metabolismo , Metilmalonil-CoA Mutasa/metabolismo , Animales , Sitios de Unión , Transporte Biológico , Catálisis , Cobamidas/química , Humanos , Metionina Adenosiltransferasa/química , Metilmalonil-CoA Mutasa/química , Methylobacterium extorquens/enzimología , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismoRESUMEN
Adenosyltransferases (ATRs) catalyze the synthesis of the reactive cobalt-carbon bond found in coenzyme B(12) or 5'-deoxyadenosylcobalamin (AdoCbl), which serves as a cofactor for a number of isomerases. The reaction involves a reductive adenosylation of cob(II)alamin in which an electron delivered by a reductase reduces cob(II)alamin to cob(I)alamin, which attacks the 5'-carbon of ATP to form AdoCbl and inorganic triphosphate. Of the three classes of ATRs found in nature, the PduO type, which is also the only one found in mammals, is the most extensively studied. The crystal structures of a number of PduO-type ATRs are available and reveal a trimeric organization with the active sites located at the subunit interfaces. We have previously demonstrated that the ATR from Methylobacterium extorquens, which supports methylmalonyl-CoA mutase activity, serves dual functions; i.e., it tailors the active AdoCbl form of the cofactor and then transfers it directly to the dependent mutase (Padovani et al. (2008) Nat. Chem. Biol. 4, 194). Only two of the three active sites in ATR are simultaneously occupied by AdoCbl. In this study, we demonstrate that binding of the substrate ATP to ATR that is fully loaded with AdoCbl leads to the ejection of 1 equivalent of the cofactor into solution. In the presence of methylmalonyl-CoA mutase and ATP, AdoCbl is transferred from ATR to the acceptor protein in a process that exhibits an approximately 3.5-fold lower K(act) for ATP compared to the one in which cofactor is released into solution. Furthermore, ATP favorably influences cofactor transfer in the forward direction by reducing the ratio of apo-methylmalonyl-CoA mutase/holo-ATR required for delivery of 1 equivalent of AdoCbl, from 4 to 1. These results lead us to propose a rotary mechanism for ATR function in which, at any given time, only two of its active sites are used for AdoCbl synthesis and where binding of ATP to the vacant site leads to the transfer of the high value AdoCbl product to the acceptor mutase.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cobamidas/biosíntesis , Cobamidas/química , Transferasas/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Escherichia coli/metabolismo , Cinética , Metilmalonil-CoA Mutasa/química , Metilmalonil-CoA Mutasa/metabolismo , Modelos Moleculares , Especificidad por Sustrato , Termodinámica , Transferasas/químicaRESUMEN
MMAB (methylmalonic aciduria type B) is a mitochondrial enzyme involved in the metabolism of vitamin B(12). It functions as the ATP:cob(I)alamin adenosyltransferase for the generation of adenosylcobalamin (AdoCbl), the cofactor of methylmalonyl-CoA mutase (MCM). Impaired MMAB activity leads to the inherited disorder methylmalonic aciduria and is responsible for the cblB complementation group. In this study, the effects on substrate binding of two catalytically inactive patient mutations, R190H and R186W, were investigated using intrinsic fluorescence quenching of MMAB as a measure of ligand-binding. We report the dissociation constant (K(d)) of wild-type MMAB for HOCbl is 51 microM and for ATP is 365 microM and show that cobalamin enhances the affinity of MMAB for ATP, while ATP does not show detectable effects on cobalamin binding. We confirm that residue Arg190 plays a role in the formation of the ATP-binding site as described previously [H.L. Schubert, C.P. Hill, Structure of ATP-bound human ATP:cobalamin adenosyltransferase, Biochemistry 45 (2006) 15188-15196]. Unexpectedly, mutation R186W does not disrupt the binding of HOCbl to MMAB as predicted; instead, both R190H and R186W significantly disrupt the affinity between MMAB and AdoCbl. We surmise that these two residues may be critical for the transfer of the 5'-deoxyadenosyl group from ATP to cob(I)alamin, possibly by contributing to the precise positioning of the two substrates to permit catalysis to occur. Characterization of ligand-binding by MMAB provides insight into the mechanism of cobalamin adenosylation and the effect of patient mutations in the inherited disorder.
Asunto(s)
Adenosina Trifosfato/metabolismo , Transferasas Alquil y Aril/genética , Errores Innatos del Metabolismo/enzimología , Mutación , Adenosina Trifosfato/química , Transferasas Alquil y Aril/metabolismo , Sitios de Unión , Catálisis , Humanos , Ligandos , Espectrometría de Fluorescencia , Vitamina B 12/metabolismoRESUMEN
Persulfides of cysteine (CysSSH), glutathione (GSSH) or N-methoxycarbonyl-penicillamine (NAcPenSSH) react with the ferric form of myoglobin (metMb(iii)) to yield the oxy-ferrous (oxyMb(ii)) or deoxy-ferrous (deoxyMb(ii)) forms of myoglobin under aerobic or anaerobic conditions, respectively. Under aerobic conditions, CysSSH and NAcPenSSH react with the hypervalent form of myoglobin (ferrylMb(iv)) to yield oxyMb(ii) as the final product with the formation of metMb(iii) as an intermediate. CysSSH and NAcPenSSH coordinate the ferric form of N-acetylated microperoxidase (NAcMP11(iii)) to yield the disulfanido complex NAcMP11(iii)(NAcPenSS), as shown by UV-vis and EPR spectroscopy. Experiments carried out with various NAcMP11 derivatives demonstrate a redox equilibrium between the ferric/ferrous forms of the heme and the polysulfides/persulfides couple. Our results suggest that persulfides possess uncommon redox properties, analogous to that of dihydrolipoic acid.
Asunto(s)
Coenzimas/metabolismo , Hemo/metabolismo , Mioglobina/metabolismo , Peroxidasas/metabolismo , Sulfuros/metabolismo , Acetilación , Hierro/metabolismo , Oxidación-Reducción , Solubilidad , Agua/químicaRESUMEN
Accumulating evidence suggests that abnormal levels of homocysteine are associated with vascular dysfunctions, cancer cell proliferation and various neurodegenerative diseases. With respect to the latter, a perturbation of transition metal homeostasis and an inhibition of catalase bioactivity have been reported. Herein, we report on some of the molecular bases for the cellular toxicity of homocysteine and demonstrate that it induces the formation of sulfcatalase, an irreversible inactive state of the enzyme, without the intervention of hydrogen sulfide. Initially, homocysteine reacts with native catalase and/or redox-active transition metal ions to generate thiyl radicals that mediate compound II formation, a temporarily inactive state of the enzyme. Then, the ferryl centre of compound II intervenes into the unprecedented S-oxygenation of homocysteine to engender the corresponding sulfenic acid species that further participates into the prosthetic heme modification through the formation of an unusual Fe(II) sulfonium. In addition, our ex cellulo studies performed on cancer cells, models of neurodegenerative diseases and ulcerative colitis suggest the likelihood of this scenario in a subset of cancer cells, as well as in a cellular model of Parkinson's disease. Our findings expand the repertoire of heme modifications promoted by biological compounds and point out another deleterious trait of disturbed homocysteine levels that could participate in the aetiology of these diseases.
Asunto(s)
Catalasa/metabolismo , Hemo/análogos & derivados , Homocisteína/metabolismo , Neoplasias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Oxígeno/metabolismo , Animales , Catalasa/antagonistas & inhibidores , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Activación Enzimática/efectos de los fármacos , Hemo/química , Hemo/metabolismo , Sulfuro de Hidrógeno/metabolismo , Hierro/metabolismo , Masculino , Espectrometría de Masas , Ratones Endogámicos C57BL , Neoplasias/patología , Oxidación-Reducción , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Small GTP-binding proteins of the Arf family (Arf GTPases) interact with multiple cellular partners and with membranes to regulate intracellular traffic and organelle structure. Understanding the underlying molecular mechanisms requires in vitro biochemical assays to test for regulations and functions. Such assays should use proteins in their cellular form, which carry a myristoyl lipid attached in N-terminus. N-myristoylation of recombinant Arf GTPases can be achieved by co-expression in E. coli with a eukaryotic N-myristoyl transferase. However, purifying myristoylated Arf GTPases is difficult and has a poor overall yield. Here we show that human Arf6 can be N-myristoylated in vitro by recombinant N-myristoyl transferases from different eukaryotic species. The catalytic efficiency depended strongly on the guanine nucleotide state and was highest for Arf6-GTP. Large-scale production of highly pure N-myristoylated Arf6 could be achieved, which was fully functional for liposome-binding and EFA6-stimulated nucleotide exchange assays. This establishes in vitro myristoylation as a novel and simple method that could be used to produce other myristoylated Arf and Arf-like GTPases for biochemical assays.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Aciltransferasas/metabolismo , Arabidopsis/enzimología , Clonación Molecular , Ácido Mirístico/metabolismo , Saccharomyces cerevisiae/enzimología , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/aislamiento & purificación , Aciltransferasas/genética , Aciltransferasas/aislamiento & purificación , Arabidopsis/genética , Escherichia coli/genética , Guanosina Trifosfato/metabolismo , Humanos , Liposomas/metabolismo , Modelos Moleculares , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaAsunto(s)
Homocisteína/sangre , Neoplasias/sangre , Enfermedades Neurodegenerativas/sangre , Homocisteína/metabolismo , Humanos , Hiperhomocisteinemia/sangre , Hiperhomocisteinemia/complicaciones , Hiperhomocisteinemia/metabolismo , Redes y Vías Metabólicas , Metionina/metabolismo , Neoplasias/etiología , Neoplasias/metabolismo , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Factores de RiesgoRESUMEN
The recognition of eight discrete genetic complementation groups among patients with inherited cobalamin disorders provided early insights into the complexity of a cofactor-processing pathway that supports only two known B(12)-dependent enzymes in mammals. With the identification of all eight genes now completed, biochemical interrogations of their functions have started and are providing novel insights into a trafficking pathway involving porters that tinker with and tailor the active cofactor forms and editors that ensure the fidelity of the cofactor loading process. The principles of sequestration and escorted delivery of a rare and reactive organometallic cofactor that are emerging from studies on B(12) might be of general relevance to other cofactor trafficking pathways.
Asunto(s)
Vitamina B 12/análisis , Vitamina B 12/metabolismo , Animales , Biocatálisis , Transporte Biológico , Humanos , Modelos MolecularesRESUMEN
In mammals, the two enzymes in the trans-sulfuration pathway, cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), are believed to be chiefly responsible for hydrogen sulfide (H2S) biogenesis. In this study, we report a detailed kinetic analysis of the human and yeast CBS-catalyzed reactions that result in H2S generation. CBS from both organisms shows a marked preference for H2S generation by beta-replacement of cysteine by homocysteine. The alternative H2S-generating reactions, i.e. beta-elimination of cysteine to generate serine or condensation of 2 mol of cysteine to generate lanthionine, are quantitatively less significant. The kinetic data were employed to simulate the turnover numbers of the various CBS-catalyzed reactions at physiologically relevant substrate concentrations. At equimolar concentrations of CBS and CSE, the simulations predict that H2S production by CBS would account for approximately 25-70% of the total H2S generated via the trans-sulfuration pathway depending on the extent of allosteric activation of CBS by S-adenosylmethionine. The relative contribution of CBS to H2S genesis is expected to decrease under hyperhomocysteinemic conditions. CBS is predicted to be virtually the sole source of lanthionine, and CSE, but not CBS, efficiently cleaves lanthionine. The insensitivity of the CBS-catalyzed H2S-generating reactions to the grade of hyperhomocysteinemia is in stark contrast to the responsiveness of CSE and suggests a previously unrecognized role for CSE in intracellular homocysteine management. Finally, our studies reveal that the profligacy of the trans-sulfuration pathway results not only in a multiplicity of H2S-yielding reactions but also yields novel thioether metabolites, thus increasing the complexity of the sulfur metabolome.
Asunto(s)
Cistationina betasintasa/química , Cistationina gamma-Liasa/química , Sulfuro de Hidrógeno/química , Alanina/análogos & derivados , Alanina/química , Cromatografía Líquida de Alta Presión , Simulación por Computador , Cisteína/química , Humanos , Cinética , Espectrometría de Masas/métodos , Modelos Biológicos , Modelos Químicos , Conformación Molecular , Serina/química , Sulfuros/química , Azufre/químicaRESUMEN
Although there is a growing recognition of the significance of hydrogen sulfide (H(2)S) as a biological signaling molecule involved in vascular and nervous system functions, its biogenesis and regulation are poorly understood. It is widely assumed that desulfhydration of cysteine is the major source of H(2)S in mammals and is catalyzed by the transsulfuration pathway enzymes, cystathionine beta-synthase and cystathionine gamma-lyase (CSE). In this study, we demonstrate that the profligacy of human CSE results in a variety of reactions that generate H(2)S from cysteine and homocysteine. The gamma-replacement reaction, which condenses two molecules of homocysteine, yields H(2)S and a novel biomarker, homolanthionine, which has been reported in urine of homocystinuric patients, whereas a beta-replacement reaction, which condenses two molecules of cysteine, generates lanthionine. Kinetic simulations at physiologically relevant concentrations of cysteine and homocysteine, reveal that the alpha,beta-elimination of cysteine accounts for approximately 70% of H(2)S generation. However, the relative importance of homocysteine-derived H(2)S increases progressively with the grade of hyperhomocysteinemia, and under conditions of severely elevated homocysteine (200 microm), the alpha,gamma-elimination and gamma-replacement reactions of homocysteine together are predicted to account for approximately 90% of H(2)S generation by CSE. Excessive H(2)S production in hyperhomocysteinemia may contribute to the associated cardiovascular pathology.
Asunto(s)
Alanina/análogos & derivados , Cistationina gamma-Liasa/metabolismo , Hiperhomocisteinemia/metabolismo , Sulfuros/química , Azufre/química , Alanina/química , Catálisis , Cisteína/química , Relación Dosis-Respuesta a Droga , Humanos , Sulfuro de Hidrógeno/química , Concentración de Iones de Hidrógeno , Cinética , Espectrometría de Masas , Modelos Químicos , Proteínas Recombinantes/química , TemperaturaRESUMEN
MeaB is an auxiliary protein that plays a crucial role in the protection and assembly of the B(12)-dependent enzyme methylmalonyl-CoA mutase. Impairments in the human homologue of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism. To explore the role of this metallochaperone, its structure was solved in the nucleotide-free form, as well as in the presence of product, GDP. MeaB is a homodimer, with each subunit containing a central alpha/beta-core G domain that is typical of the GTPase family, as well as alpha-helical extensions at the N and C termini that are not found in other metalloenzyme chaperone GTPases. The C-terminal extension appears to be essential for nucleotide-independent dimerization, and the N-terminal region is implicated in protein-protein interaction with its partner protein, methylmalonyl-CoA mutase. The structure of MeaB confirms that it is a member of the G3E family of P-loop GTPases, which contains other putative metallochaperones HypB, CooC, and UreG. Interestingly, the so-called switch regions, responsible for signal transduction following GTP hydrolysis, are found at the dimer interface of MeaB instead of being positioned at the surface of the protein where its partner protein methylmalonyl-CoA mutase should bind. This observation suggests a large conformation change of MeaB must occur between the GDP- and GTP-bound forms of this protein. Because of their high sequence homology, the missense mutations in MMAA that result in methylmalonic aciduria have been mapped onto MeaB and, in conjunction with mutagenesis data, provide possible explanations for the pathology of this disease.
Asunto(s)
Desequilibrio Ácido-Base/genética , Ácido Metilmalónico/orina , Metilmalonil-CoA Mutasa/metabolismo , Chaperonas Moleculares/genética , Mutagénesis , Errores Innatos del Metabolismo de los Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Metilmalonil-CoA Mutasa/química , Metilmalonil-CoA Mutasa/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Mutación Puntual , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Espectrometría RamanRESUMEN
Methylmalonyl-CoA mutase catalyzes the adenosylcobalamin-dependent rearrangement of (2R)-methylmalonyl-CoA to succinyl-CoA. The crystal structure of the enzyme reveals that Y243 is in van der Waals contact with the methyl group of the substrate and suggests a possible role for it in the stereochemical control of the reaction. This hypothesis was tested by designing a molecular hole by replacing the phenolic side chain of Y243 with the methyl group of alanine. The Y243A mutation lowered the catalytic efficiency >(4 x 10(4))-fold compared to wild-type enzyme, the K(M)app for the cofactor approximately 4-fold, and the cob(II)alamin concentration under steady-state turnover conditions approximately 2-fold. However, the mutation did not appear to lead to loss of the stereochemical preference for the substrate. The Y243A mutation is expected to create a cavity and should, in principle, allow accommodation of bulkier substrates. To test this, we used ethylmalonyl-CoA and allylmalonyl-CoA as alternate substrates. Surprisingly, both analogues resulted in suicidal inactivation, albeit in an O(2)-dependent and O(2)-independent fashion, respectively. The inactivation by allylmalonyl-CoA was further investigated, and revealed formation of cob(II)alamin at an approximately 1.5-fold higher rate than with wild-type mutase under single-turnover conditions. Product analysis revealed a stoichiometric mixture of 5'-deoxyadenosine, aquocobalamin, and allylmalonyl-CoA. Taken together, these results are consistent with an internal electron transfer from cob(II)alamin to the substrate analogue radical. These studies serve to emphasize the fine control exerted by Y243 in the vicinity of the substrate to minimize radical extinction in side reactions.