Production of the Neuromodulator H2S by Cystathionine β-Synthase via the Condensation of Cysteine and Homocysteine

Hydrogen sulfide (H2S) has been observed in relatively high concentrations in the mammalian brain and has been shown to act as a neuromodulator. However, there is confusion in the literature regarding the actual source of H2S production. Reactions catalyzed by the cystathionine β-synthase enzyme (CBS) are one possible source for the production of H2S. Here we show that the CBS enzyme can efficiently produce H2S via a β-replacement reaction in which cysteine is condensed with homocysteine to form cystathionine and H2S. The production of H2S by this reaction is at least 50 times more efficient than that produced by hydrolysis of cysteine alone via β-elimination. Kinetic studies demonstrate that the Km and Kcat for cysteine is 3-fold higher and 2-fold lower, respectively, than that for serine. Consistent with these data, in vitro reconstitution studies show that at physiologically relevant concentrations of serine, homocysteine, and cysteine, about 5% of the cystathionine formed is from cysteine. We also show that AdoMet stimulates this H2S producing reaction but that there is no evidence for stimulation by calcium and calmodulin as reported previously. In summary, these results confirm the ability of CBS to produce H2S, but show in contrast to prior reports that the major mechanism is via β-replacement and not cysteine hydrolysis. In addition, these studies provide a biochemical explanation for the previously inexplicable homocysteine-lowering effects of N-acetylcysteine treatments in humans. Hydrogen sulfide (H2S) has been observed in relatively high concentrations in the mammalian brain and has been shown to act as a neuromodulator. However, there is confusion in the literature regarding the actual source of H2S production. Reactions catalyzed by the cystathionine β-synthase enzyme (CBS) are one possible source for the production of H2S. Here we show that the CBS enzyme can efficiently produce H2S via a β-replacement reaction in which cysteine is condensed with homocysteine to form cystathionine and H2S. The production of H2S by this reaction is at least 50 times more efficient than that produced by hydrolysis of cysteine alone via β-elimination. Kinetic studies demonstrate that the Km and Kcat for cysteine is 3-fold higher and 2-fold lower, respectively, than that for serine. Consistent with these data, in vitro reconstitution studies show that at physiologically relevant concentrations of serine, homocysteine, and cysteine, about 5% of the cystathionine formed is from cysteine. We also show that AdoMet stimulates this H2S producing reaction but that there is no evidence for stimulation by calcium and calmodulin as reported previously. In summary, these results confirm the ability of CBS to produce H2S, but show in contrast to prior reports that the major mechanism is via β-replacement and not cysteine hydrolysis. In addition, these studies provide a biochemical explanation for the previously inexplicable homocysteine-lowering effects of N-acetylcysteine treatments in humans. Recently, there has been increased interest in endogenously produced hydrogen sulfide (H2S) as a physiologically important molecule. Relatively high concentrations of H2S have been observed in the brains of rats, humans, and cows (1Goodwin L.R. Francom D. Dieken F.P. Taylor J.D. Warenycia M.W. Reiffenstein R.J. Dowling G. J. Anal. Toxicol. 1989; 13: 105-109Crossref PubMed Scopus (241) Google Scholar, 2Warenycia M.W. Goodwin L.R. Benishin C.G. Reiffenstein R.J. Francom D.M. Taylor J.D. Dieken F.P. Biochem. Pharmacol. 1989; 38: 973-981Crossref PubMed Scopus (261) Google Scholar, 3Savage J.C. Gould D.H. J. Chromatogr. 1990; 526: 540-545Crossref PubMed Scopus (151) Google Scholar). At physiological concentrations it has been shown that H2S enhances N-methyl-d-asparate receptor-mediated response and can modify long term potentiation (4Abe K. Kimura H. J. Neurosci. 1996; 16: 1066-1071Crossref PubMed Google Scholar, 5Kimura H. Biochem. Biophys. Res. Commun. 2000; 267: 129-133Crossref PubMed Scopus (300) Google Scholar, 6Kimura H. Mol. Neurobiol. 2002; 26: 13-19Crossref PubMed Google Scholar). H2S also inhibits smooth muscle cell proliferation via the mitogen-activated protein kinase pathway and protects neurons against oxidative stress (7Kimura Y. Kimura H. FASEB J. 2004; 18: 1165-1167Crossref PubMed Scopus (720) Google Scholar). H2S also appears to have an effect on the cardiovascular system, acting as a vasorelaxant by increasing potassium-ATP channel currents (8Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1600) Google Scholar). Taken together, these observations suggest that endogenously produced H2S is an important regulatory molecule in humans. How is endogenous H2S produced? Potential sources are alternative reactions catalyzed by the enzyme cystathionine β-synthase (CBS) 1The abbreviations used are: CBS, cystathionine β-synthase enzyme; PLP, pyridoxal phosphate; PBS, phosphate-buffered saline; GST, glutathione S-transferase; Bicine, N,N-bis(2-hydroxyethyl)glycine; AdoMet, S-adenosylmethionine; aa, amino acids; CGL, cystathionine γ-lyase.1The abbreviations used are: CBS, cystathionine β-synthase enzyme; PLP, pyridoxal phosphate; PBS, phosphate-buffered saline; GST, glutathione S-transferase; Bicine, N,N-bis(2-hydroxyethyl)glycine; AdoMet, S-adenosylmethionine; aa, amino acids; CGL, cystathionine γ-lyase. (6Kimura H. Mol. Neurobiol. 2002; 26: 13-19Crossref PubMed Google Scholar). The normal cellular function of CBS is to catalyze the condensation of serine with homocysteine to form cystathionine and water, a key reaction in the transsulfuration pathway. CBS uses pyridoxal phosphate (PLP) as a co-factor and is a member of the β-family or fold type II of PLP containing enzymes. Enzymes in this family characteristically have the ability to catalyze β-replacement and β-elimination reactions from a variety of different substrates (9Miles E.W. Dolphin D. Poulson D. Avramovic O. Pyridoxal Phosphate: Chemical, Biochemical and Medical Aspects, Part B. John Wiley and Sons, New York1986: 235-310Google Scholar). There are two potential mechanisms through which CBS could produce H2S. First, CBS could catalyze the production of H2S from cysteine by a β-elimination or an α,β-elimination reaction (Fig. 1, Alternate Reactions 3 and 4, respectively). This type of reaction has been reported to occur with CBS isolated from mouse brain and from CBS present in rat liver and kidney extracts (6Kimura H. Mol. Neurobiol. 2002; 26: 13-19Crossref PubMed Google Scholar, 10Stipanuk M.H. Beck P.W. Biochem. J. 1982; 206: 267-277Crossref PubMed Scopus (599) Google Scholar). An alternative source for H2S production would involve a β-replacement reaction. Using this mechanism CBS can produce H2S from the reaction of l-cysteine and 2-mercaptoethanol to form S-hydroxyethyl-l-cysteine and H2S (Fig. 1, Alternate Reaction 2) (11Jhee K.H. McPhie P. Miles E.W. Biochemistry. 2000; 39: 10548-10556Crossref PubMed Scopus (79) Google Scholar). While this reaction would not be expected to occur in vivo, a similar β-replacement reaction could occur by the condensation of homocysteine with cysteine (Fig. 1, Alternate Reaction 1). This potential reaction is interesting because it would also be an alternative method for metabolizing homocysteine. Elevated plasma homocysteine levels have been linked to a variety of human diseases, including heart attack, stroke, Alzheimer disease, and osteoporosis (12Refsum H. Smith A.D. Ueland P.M. Nexo E. Clarke R. McPartlin J. Johnston C. Engbaek F. Schneede J. McPartlin C. Scott J.M. Clin. Chem. 2004; 50: 3-32Crossref PubMed Scopus (872) Google Scholar, 13Seshadri S. Beiser A. Selhub J. Jacques P.F. Rosenberg I.H. D'Agostino R.B. Wilson P.W. Wolf P.A. N. Engl. J. Med. 2002; 346: 476-483Crossref PubMed Scopus (2783) Google Scholar, 14van Meurs J.B. Dhonukshe-Rutten R.A. Pluijm S.M. van der Klift M. de Jonge R. Lindemans J. de Groot L.C. Hofman A. Witteman J.C. van Leeuwen J.P. Breteler M.M. Lips P. Pols H.A. Uitterlinden A.G. N. Engl. J. Med. 2004; 350: 2033-2041Crossref PubMed Scopus (625) Google Scholar, 15McLean R.R. Jacques P.F. Selhub J. Tucker K.L. Samelson E.J. Broe K.E. Hannan M.T. Cupples L.A. Kiel D.P. N. Engl. J. Med. 2004; 350: 2042-2049Crossref PubMed Scopus (492) Google Scholar). Since CBS is a key regulator of homocysteine, it is possible that this alternative reaction may have clinical relevance. In this paper we report the characterization of the biochemical and kinetic properties of human CBS in catalyzing various H2S-producing reactions. We find that human CBS can efficiently catalyze the formation of H2S via the condensation of homocysteine with cysteine and that this reaction is likely to occur in vivo. CBS Expression Systems—Two expression systems were used to produce human CBS. For the yeast system we used a yeast strain (WY218) that was deleted for endogenous yeast CBS (CYS4) and deleted for yeast O-acetylserine/O-acetylhomoserine sulfhydrylase. Extracts from WY218 exhibit no CBS activity and have no ability to form H2S (see Fig. 2, lane 1). Into this strain was transformed a plasmid expressing either wild-type human CBS or a truncated human CBS (amino acids 1–409) (16Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (106) Google Scholar). Total yeast extracts were made as described previously (17Shan X. Dunbrack Jr., R.L. Christopher S.A. Kruger W.D. Hum. Mol. Genet. 2001; 10: 635-643Crossref PubMed Google Scholar). For bacterial expression, Escherichia coli BL21 (DE3) containing pGEX-CBS (16Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (106) Google Scholar) were grown to an A600 of 0.6 in LB medium at 37 °C. isopropyl 1-thio-β-d-galactopyranoside was added to a final concentration of 0.05 mm to induce the expression of fusion protein at 20 °C. The cells were resuspended in PBS containing 10 mm dithiothreitol, 100 mm MgCl2, 0.5 mg/ml lysozyme, 2 units/ml DNase, and 0.86 mg/ml protease inhibitor mixture (Sigma) for 1 h at 4 °C and then lysed by freeze-thawing two times. The lysates were incubated at 4 °C for an additional 30 min, briefly sonicated on ice to reduce viscosity, and centrifuged, and the clarified supernatants containing fusion proteins were filtered (0.2-μm filter) and applied to a GSTrap column (Amersham Biosciences) that was connected to a Bio-Rad Biologic Duo Flow FPLC equilibrated with PBS (pH 7.3) and 5 mm dithiothreitol. After being washed with 20 column volumes of the same buffer, the column was filled with thrombin protease, sealed, and incubated at room temperature for 16 h. Cleaved protein was eluted using 20 ml of PBS, and bound GST and uncleaved GST fusion proteins were eluted with 20 mm reduced glutathione in Tris-HCl (pH 8.0). Analysis of the purified human CBS protein by SDS-PAGE and Coomassie Brilliant Blue staining indicated that the protein was >95% pure. CBS Enzyme Activity Assays—We used three different assays to assess CBS enzyme activity. Assays involving crude extracts were first dialyzed overnight against 50 mm Na/Bicine (pH 8.6) buffer containing 50 μm PLP in a Slide-A-Lyzer mini-dialysis unit (Pierce). For the native gel assays, H2S production was assayed by reaction with Pb-acetate using a modified previously described procedure (11Jhee K.H. McPhie P. Miles E.W. Biochemistry. 2000; 39: 10548-10556Crossref PubMed Scopus (79) Google Scholar, 18Willhardt I. Wiederanders B. Anal. Biochem. 1975; 63: 263-266Crossref PubMed Scopus (16) Google Scholar). Sixty or 100 μg of yeast extract was loaded on the 8% native Tris-glycine gels (Novex) at 4 °C. After gel electrophoresis was finished, active protein bands in native gels were detected by soaking the gel (8.5 × 7.5 cm) in 50 ml of the reaction assay mixtures for several hours to overnight at room temperature. The reaction mixture contained 200 mm Na/Bicine (pH 8.6), 50 μm PLP, 0.25 mg/ml bovine serum albumin, 0.4 mm lead acetate, and substrates: reaction 1 substrates (10 mm l-cysteine, 10 mm l-homocysteine), reaction 2 substrates (10 mm l-cysteine, 10 mm 2-mercaptoethanol), reaction 3 and 4 substrate (10 mm l-cysteine), respectively. For production of H2S from purified CBS, a spectrophotometric assay was used. The reaction of H2S with lead acetate to form lead sulfide was monitored continuously by the increase in absorbance at 390 nm in a Hewlett Packard 8453 diode array spectrophotometer thermostatted at 37 °C. Reaction mixture (1 ml) contained 200 ng of purified CBS using the reaction conditions described above. Quantitation of H2S production was performed using a calibration curve obtained by comparing A390 with cystathionine production using a Biochrom 30 amino acid analyzer. For kinetic and other studies, enzyme activities were measured using standard reaction containing 100 mm Na/Bicine (pH 8.6), 200 μm PLP, 1 mm tris(2-carboxyethyl)phosphine, 100 μm AdoMet, 0.25 mg/ml bovine serum albumin, and 10 mm l-homocysteine. Total reaction volume was 50 μl. The concentrations of co-substrates serine or cysteine were between 0 and 40 mm. Reactions were carried out at 37 °C for 1 h, and the cystathionine produced was measured by an amino acid analyzer (Biochrom 30). The kinetic parameters were determined using EnzKinetics software. Determination of Mouse Liver Amino Acid Concentrations—Livers from C57BL6 animals maintained on standard mouse chow (LabDiet, 5013) were harvested, weighed, then extracted as described previously (19Wang L. Jhee K.H. Hua X. DiBello P.M. Jacobsen D.W. Kruger W.D. Circ. Res. 2004; 94: 1318-1324Crossref PubMed Scopus (81) Google Scholar). The volume of the total extract was then carefully measured, and 50 ml were analyzed using a Biochrom 30 amino acid analyzer. Amino Acid Analysis—Samples for amino acid analysis were first processed by addition of dithiothreitol to a final concentration of 1.2% and incubation on ice for 10 min. This was followed by addition of sulfocylic acid to 5% and centrifugation at 12,000 × g for 10 min. The samples were then loaded on the Biochrom 30 using an autoloader. Quantitation was performed by calibrating the peak heights to a standard in which the amounts of cysteine, serine, and homocysteine were known. The program EZ Chrom Elite was used to analyze the data. H2S Production by Human CBS in Saccharomyces cerevisiae—To identify whether human CBS had H2S-forming activity, we measured H2S formation using a gel activity assay. In this method, yeast extracts are separated on native gels, exposed to various substrates, and then assessed for H2S formation in situ (see “Materials and Methods”). We examined H2S formation from either 10 mm l-cysteine alone, 10 mm l-cysteine with 10 mm l-homocysteine, or 10 mm l-cysteine and 10 mm 2-mercaptoethanol. The yeast strain we used (WY218) was deleted for endogenous yeast CBS and contained either a control plasmid, a plasmid expressing wild-type human CBS (aa 1–551), or one expressing a truncated human CBS (aa 1–409) lacking the C-terminal regulatory domain. The truncated form of CBS has been shown to be hyperactive and not responsive to allosteric regulation by AdoMet (16Shan X. Kruger W.D. Nat. Genet. 1998; 19: 91-93Crossref PubMed Scopus (106) Google Scholar). As shown in Fig. 2, both the full-length and truncated form of the enzyme have significant H2S forming ability when cysteine is combined with either homocysteine or β-mercaptoethanol. However, neither full-length nor truncated CBS has significant H2S forming ability when only cysteine is present. results show that human CBS is more active at producing β-replacement reaction then by a β-elimination reaction. AdoMet is an allosteric of CBS that stimulates CBS activity by the of the C-terminal (17Shan X. Dunbrack Jr., R.L. Christopher S.A. Kruger W.D. Hum. Mol. Genet. 2001; 10: 635-643Crossref PubMed Google Scholar). As we that addition of AdoMet H2S formation from the wild-type enzyme (Fig. 2) but not the truncated This that the regulation of the H2S forming β-replacement reactions is similar to that of the reaction. We also examined the H2S forming ability of human CBS purified from E. In this reactions were carried out in and lead was determined using a As shown in Fig. the purified enzyme significant levels of H2S when either 10 mm or 10 mm were combined with 10 mm However, when 10 mm l-cysteine was incubated in the of a we no levels of H2S results confirm that CBS H2S through condensation of cysteine with homocysteine other via β-replacement than through cysteine hydrolysis and an reaction. Kinetic of Human the of of of these We determined the Km and for both and l-cysteine conditions in which was present at 10 mm in the reaction We that the Km of cysteine with serine was higher and the The of is about that physiological conditions serine would be in to serine kinetic in a examined the of either cysteine or serine in the formation of cystathionine in an in vitro assay. serine, cysteine, or both were added to 200 ng of purified CBS and incubated for 1 h at 37 °C. cystathionine formation and were measured using an amino acid analyzer (Fig. At high concentrations (10 of serine, cysteine, and homocysteine, cysteine was used to of the serine was used to As we the concentration of substrates to 1 the of cystathionine formed from cysteine to At the concentration only of the cystathionine from cysteine. are with the kinetic 3-fold in and show that the of cysteine to serine used in cystathionine formation on the concentration of the substrates present in a In of the literature we for the concentrations of cysteine, serine, and homocysteine in mammalian D. J. Chem. PubMed Google Scholar, E. Amino Acid B. Scholar). we determined the concentrations of these amino acids in mouse liver Mouse were weighed, and then analyzed for amino acid using an amino acid analyzer. This to the amino acid of was in the we determined the of mouse and The in the of in the and this was then used as the in Using this we that in mouse liver the concentrations of serine, cysteine, and homocysteine are and respectively. In an in vitro reaction using these concentrations of substrates, we determined that about 5% of the cystathionine formed in a mouse liver was from cysteine (Fig. of and on H2S-producing Enzyme has been previously reported that CBS a calmodulin and that the hydrolysis of cysteine is by calcium and (6Kimura H. Mol. Neurobiol. 2002; 26: 13-19Crossref PubMed Google Scholar). We to effect of and or calcium addition on CBS activity from mouse liver purified human CBS produced in E. or from yeast extracts expressing human CBS not Since the reported by Kimura were on mouse we also examined mouse brain extracts for stimulation by and we to stimulation of CBS activity in the of not studies show that CBS is not by The of this was to the that CBS may have in the endogenous production of H2S in vivo. in the of has that H2S can by of the N-methyl-d-asparate (4Abe K. Kimura H. J. Neurosci. 1996; 16: 1066-1071Crossref PubMed Google Scholar, 5Kimura H. Biochem. Biophys. Res. Commun. 2000; 267: 129-133Crossref PubMed Scopus (300) Google Scholar, 6Kimura H. Mol. Neurobiol. 2002; 26: 13-19Crossref PubMed Google Scholar). However, there has confusion as to the source of H2S in vivo. of the literature on endogenous H2S that H2S is formed from the hydrolysis of cysteine by the of cystathionine The the that H2S is produced by CBS. However, the mechanism for production is not by the hydrolysis of cysteine but by a β-replacement reaction homocysteine and cysteine. This reaction is to the endogenous β-replacement reaction involving homocysteine and serine, that cysteine is for serine, in the formation of H2S of This the is by addition of we not observed evidence for the stimulation of CBS by (6Kimura H. Mol. Neurobiol. 2002; 26: 13-19Crossref PubMed Google Scholar). are in with it is with from the H2S formation from purified serine from and Biophys. PubMed Scopus Google Scholar) that liver serine had a increase in H2S-producing activity when cysteine and homocysteine were added together, with cysteine by by this same that the enzyme as liver serine was in similar to purified rat liver cystathionine and it was that these were in the same enzyme I.H. Biophys. PubMed Scopus Google Scholar). The this There have been other reports in the literature that transsulfuration cystathionine may be important in H2S production in M.H. Beck P.W. Biochem. J. 1982; 206: 267-277Crossref PubMed Scopus (599) Google Scholar, S. M. Biochem. 2002; PubMed Scopus Google but not that We not evidence of significant H2S formation in yeast extracts using gel assay when only cysteine was yeast has significant levels of activity S. M. Biochem. 2002; PubMed Scopus Google Scholar). we to H2S formation in the extracts of mouse of animals deleted for endogenous CBS. Jhee and W. D. data. The evidence that can produce H2S from in which was shown to H2S production in crude liver extracts M.H. Beck P.W. Biochem. J. 1982; 206: 267-277Crossref PubMed Scopus (599) Google Scholar). possible explanation for this is that in the of a crude rat liver extract the may be other in production or increased of H2S. suggest that the production of H2S from cysteine and homocysteine occur in vivo. We that wild-type human CBS has a Km for l-cysteine of about 3-fold higher than the Km for serine. We also that in three substrate reactions homocysteine, and a but significant of the cystathionine produced from cysteine. the substrates were added at physiological concentrations determined in mouse we that about 5% of the cystathionine produced from cysteine. This is with the that H2S levels are at least an of than cysteine, homocysteine, or levels in the brain K. K. Kimura H. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar). may also have clinical relevance. H2S levels have been shown to be in the brains of Alzheimer and in the of K. K. Kimura H. Biochem. Biophys. Res. Commun. 2002; PubMed Scopus Google Scholar). these levels of H2S are to be in human disease, it may be possible to increase H2S and homocysteine levels by increasing the concentration of cysteine in In it has been shown that of N-acetylcysteine can plasma homocysteine levels in M.T. Clin. Chem. Med. 2002; PubMed Scopus Google Scholar, A. Pharmacol. Toxicol. 2000; PubMed Google Scholar, O. G. A. P. B. 1996; PubMed Scopus Google Scholar). The likely for this effect is that N-acetylcysteine is to cysteine increasing the concentration of cysteine and the of homocysteine to This would also be expected to increase the production of H2S. In summary, the the potential of CBS in the production of H2S and the of homocysteine and the that CBS may a key in We and for of this We also the of the at