Assay methods for H2S biogenesis and catabolism enzymes.

H2S is produced from sulfur-containing amino acids, cysteine and homocysteine, or a catabolite, 3-mercaptopyruvate, by three known enzymes: cystathionine ß-synthase, γ-cystathionase, and 3-mercaptopyruvate sulfurtransferase. Of these, the first two enzymes reside in the cytoplasm and comprise the transsulfuration pathway, while the third enzyme is found both in the cytoplasm and in the mitochondrion. The following mitochondrial enzymes oxidize H2S: sulfide quinone oxidoreductase, sulfur dioxygenase, rhodanese, and sulfite oxidase. The products of the sulfide oxidation pathway are thiosulfate and sulfate. Assays for enzymes involved in the production and oxidative clearance of sulfide to thiosulfate are described in this chapter.

Structure and kinetic analysis of H2S production by human mercaptopyruvate sulfurtransferase.

Mercaptopyruvate sulfurtransferase (MST) is a source of endogenous H2S, a gaseous signaling molecule implicated in a wide range of physiological processes. The contribution of MST versus the other two H2S generators, cystathionine ß-synthase and γ-cystathionase, has been difficult to evaluate because many studies on MST have been conducted at high pH and have used varied reaction conditions. In this study, we have expressed, purified, and crystallized human MST in the presence of the substrate 3-mercaptopyruvate (3-MP). The kinetics of H2S production by MST from 3-MP was studied at pH 7.4 in the presence of various physiological persulfide acceptors: cysteine, dihydrolipoic acid, glutathione, homocysteine, and thioredoxin, and in the presence of cyanide. The crystal structure of MST reveals a mixture of the product complex containing pyruvate and an active site cysteine persulfide (Cys(248)-SSH) and a nonproductive intermediate in which 3-MP is covalently linked via a disulfide bond to an active site cysteine. The crystal structure analysis allows us to propose a detailed mechanism for MST in which an Asp-His-Ser catalytic triad is positioned to activate the nucleophilic cysteine residue and participate in general acid-base chemistry, whereas our kinetic analysis indicates that thioredoxin is likely to be the major physiological persulfide acceptor for MST.

Relative contributions of cystathionine beta-synthase and gamma-cystathionase to H2S biogenesis via alternative trans-sulfuration reactions.

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.

H2S biogenesis by human cystathionine gamma-lyase leads to the novel sulfur metabolites lanthionine and homolanthionine and is responsive to the grade of hyperhomocysteinemia.

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.

A dithio-coupled kinase and ATPase assay.

Kinases and ATPases produce adenosine diphosphate (ADP) as a common product, so an assay that detects ADP would provide a universal means for activity-based screening of enzymes in these families. Because it is known that most kinases accept ATPbetaS (sulfur on the beta-phosphorous) as a substrate in place of adenosine triphosphate (ATP), the authors have developed a continuous assay using this substrate, with detection of the ADPbetaS product using dithio reagents. Such an assay is possible because dithio groups react selectively with ADPbetaS and not with ATPbetaS. Thiol detection was done using both Ellman's reagent (DTNB) and a recently developed fluorescent dithio reagent, DSSA. Therefore, the assay can be run in both absorbance and fluorescence detection modes. The assay was used to perform steady-state kinetic analyses of both hexokinase and myosin ATPase. It was also used to demonstrate the diastereoselectivity of hexokinase (R) and myosin ATPase (S) for the isomers of ATPbetaS, consistent with previous results. When run in fluorescence mode using a plate reader, an average Z' value of 0.54 was obtained, suggesting the assay is appropriate for high-throughput screening.

Fluorescence-based detection of thiols in vitro and in vivo using dithiol probes.

Thiols play a central role in maintaining biological homeostasis. Their levels can change dramatically in response to oxidative stress associated with toxic insults, bacterial infection, and disease. Therefore, a reagent that can monitor thiol levels both in vitro and in vivo would be useful for assays and as a biomarker. Such a reagent should (i) be selective for thiols, (ii) be able to penetrate cell walls, and (iii) have a low reduction potential so as not to create oxidative stress in a cell. We have developed such a fluorescent reagent (DSSA) based on a dithiol linker: (i) the use of a dithiol linker makes it selective for thiols; (ii) the use of fluorophores that populate neutral states at physiological pH improves cell wall penetration; and (iii) because of the reagent's low reduction potential (-0.60 V), it will not stress cells oxidatively. For example, 5 microM of reagent is responsive to changes in glutathione levels in the physiologically relevant range of 1 to 10mM, yet this would oxidize less than 1% of cellular glutathione. In Escherichia coli, decreased thiol levels were detected in cells deficient in glutathione synthesis. In zebrafish embryos, the DSSA reagent permitted detection of unusually high thiol levels in the zebrafish chorion.