Production of the neuromodulator H2S by cystathionine beta-synthase via the

condensation of cysteine and homocysteine

Xulin Chen‡¶, Kwang-Hwan Jhee‡§¶, and Warren D. Kruger‡**

From the ‡Division of Population Science, Fox Chase Cancer Center, Philadelphia PA, USA and

§Department of Applied Chemistry, Kumoh National Institute of Technology, Gumi-city, Korea

¶ Authors contributed equally to this work

**To whom correspondence should be addressed: Fox Chase Cancer Center, 333 Cottman

Avenue, Philadelphia, PA 19111. Tel: 215-728-3030; Fax: 215-214-1623; E-mail:

warren.kruger@fccc.edu

khjhee@mail.kumoh.ac.kr (K.H. Jhee), Xulen.Chen@fccc.edu (X. Chen)

JBC Papers in Press. Published on November 1, 2004 as Manuscript C400481200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Abstract

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 beta-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 b-replacement reaction in

which cysteine is condensed with homocysteine to form cystathionine and H2S. The production

of H2S by this reaction is at least fifty-times more efficient than that produced by hydrolysis of

cysteine alone via b-elimination. Kinetic studies demonstrate that the Km and Kcat for cysteine is

three-fold higher and two-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 previously reported. 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 b-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.

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Introduction

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 (1-3). At physiological concentrations it has

been shown that H2S enhances N-methyl-D-asparate (NMDA) receptor-mediated response and

can modify long term potentiation (4-6). H2S also inhibits smooth muscle cell proliferation via

the MAPK pathway and protects neurons against oxidative stress (7). H2S also appears to have

an effect on the cardiovascular system, acting as a vasorelaxant by increasing potassium-ATP

channel currents (8). 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 beta-synthase (CBS) (6). 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 b-family or Fold type II of PLP containing enzymes. Enzymes in this family

characteristically have the ability to catalyze b-replacement and b-elimination reactions from a

variety of different substrates (9).

There are two potential mechanisms through which CBS could produce H2S. First, CBS

could catalyze the production of H2S from cysteine by a b-elimination or an a,b-elimination

reaction (Figure 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 (6,10). An alternative source for H2S production would involve a b-replacement

reaction. Using this mechanism CBS can produce H2S from the reaction of L-cysteine and 2-

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mercaptoethanol to form S-hydroxyethyl-L-cysteine and H2S (Figure 1, alternate reaction 2)

(11). While this reaction would not be expected to occur in vivo, a similar b-replacement

reaction could occur by the condensation of homocysteine with cysteine (Figure 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’s disease and osteoporosis

(12-15). 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.

Methods

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 Figure 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) (16). Total yeast extracts were made as previously described (17).

For bacterial expression, E. coli BL21 (DE3) containing pGEX-CBS (16) were grown to an

O.D. of 0.6 in LB media at 37°C. IPTG 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

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DTT, 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-mm 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 DTT. 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 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 mM 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 (11,18). Sixty or 100 mg 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 cm ¥ 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 mM PLP, 0.25 mg/ml bovine serum albumin, 0.4 mM

Pb-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.

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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 OD390 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, pH8.6, 200 mM PLP, 1 mM TCEP, 100 mM AdoMet, 0.25 mg/ml

BSA and 10 mM L-homocysteine. Total reaction volume was 50 ml. The concentrations of co-

substrates serine or cysteine were between 0 and 40 mM. Reactions were carried out at 37°C for

1 hour, and the cystathionine produced was measured by amino acid analyzer (Biochrom 30).

The kinetic parameters were determined using software-EnzKinetics.

Determination of Mouse Liver Amino Acid Concentrations—Livers from C57BL6 animals

maintained on standard mouse chow (LabDiet, #5013) were harvested, weighed, then extracted

as previously described (19). 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 minutes. This was

followed by addition of sulfocylic acid to 5% and centrifugation at 12,000g for 10 minutes. 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.

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Results

H2S Production by Human CBS in S. 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 (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 (aa1-551), or one

expressing a truncated human CBS (aa1-409) 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 (16). As shown in Figure 2, both the full-length and truncated form of the

enzyme have significant H2S forming ability when cysteine is combined with either

homocysteine or b-mercaptoethanol. However, neither full-length nor truncated CBS has

significant H2S forming ability when only cysteine is present. These results show that human

CBS is much more active at producing H2S by a b-replacement reaction then by a b-elimination

reaction.

AdoMet is an allosteric effector of CBS that stimulates CBS activity by relieving the

inhibition of the C-terminal domain (17). As expected, we found that addition of AdoMet

stimulated H2S formation from the wild-type enzyme (Figure 2), but not the truncated enzyme.

This result suggests that the regulation of the H2S forming b-replacement reactions is similar to

that of the canonical reaction.

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We also examined the H2S forming ability of recombinant human CBS purified from E.

coli. In this experiment, reactions were carried out in solution and lead sulfate accumulation was

determined using a spectrophotometer. As shown in Figure 2b, the purified enzyme produces

significant levels of H2S when either 10 mM L-homocysteine or 10 mM b-mercaptoethanol were

combined with 10 mM L-cysteine. However, when 10 mM L-cysteine was incubated in the

absence of a co-substrate we saw no detectable levels of H2S formation. These results confirm

that CBS produces H2S through condensation of cysteine with homocysteine (or some other

substrate) via b-replacement rather than through cysteine hydrolysis and an elimination reaction.

Kinetic Studies of Purified Human CBS—We next compared the relative efficiency of

utilization of each of these substrates. We determined the Km and Vmax for both L-serine and L-

cysteine under conditions in which L-homocysteine was present at 10 mM in the reaction

mixture. We found that the Km of cysteine compared to serine was 3.5-fold higher (Table 1) and

the Vmax 2.3-fold reduced. The ratio of Kcat/Km is about eight-fold decreased, suggesting that

under physiological conditions serine would be utilized in preference to cysteine.

Competition Studies—We next examined the utilization of either cysteine or serine in the

formation of cystathionine in an in vitro competition assay. Either serine, cysteine or both were

added to 200 ng of purified recombinant CBS and incubated for one hour at 370C. Subsequently,

cystathionine formation and serine/cysteine usage were measured using an amino acid analyzer

(Figure 3). At high concentrations (10 mM) of serine, cysteine and homocysteine, cysteine was

used to make 44% of the cystathionine, while serine was used to make 56%. As we decreased

the concentration of substrates to 1 mM, the amount of cystathionine formed from cysteine

decreased to 35%. At the lowest concentration tested (0.1 mM) only 23% of the cystathionine

came from cysteine. These data are consistent with the kinetic data (a three-fold difference in

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Km) and show that the ratio of cysteine to serine used in cystathionine formation depends greatly

on the concentration of the substrates present in a tissue.

In examination of the literature we found widely varying references for the concentrations

of cysteine, serine and homocysteine in mammalian livers (20,21). Therefore, we determined the

concentrations of these amino acids in mouse liver ourselves. Mouse livers were weighed,

extracted and then analyzed for free amino acid content using an amino acid analyzer. This

allowed us to determine the free amino acid content per milligram of liver. In order to estimate

how much water was in the tissue, we determined the weight of mouse livers before and after

desiccation. The difference in weight indicates the amount of water in the sample, and this

number was then used as the denominator in our calculations. Using this procedure, we

estimated that in mouse liver the concentrations of serine, cysteine, and homocysteine are 0.72

mM, 0.47 mM, and 0.58 mM, 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

derived from cysteine (Figure 3, right hand panel).

No Effects of Calcium and Calmodulin on H2S Producing Enzyme Activity—It has been

previously reported that CBS contains a calmodulin (CaM) binding motif and that the hydrolysis

of cysteine is regulated by calcium and CaM (6). We failed to see any effect of CaM and or

calcium addition on CBS activity from mouse liver extract, purified recombinant human CBS

produced in E. coli, or from yeast extracts expressing human CBS (data not shown). Since the

experiments reported by Kimura were done on mouse brain, we also examined mouse brain

extracts for stimulation by Ca2+ and CaM. Again, we failed to see any stimulation of CBS

activity in the presence of Ca2+/CaM (data not shown). These studies show that CBS is not

regulated by Ca/CaM.

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Discussion

The goal of this work was to clarify the role that CBS may have in the endogenous

production of H2S in vivo. Work in the field of molecular neurology has clearly established that

H2S can modulate neuronal signals by modulating signaling of the NMDA receptor (4-6).

However, there has existed confusion as to the source of H2S in vivo. Most of the literature on

endogenous H2S suggests that H2S is formed primarily from the hydrolysis of cysteine by the

action of cystathionine beta-synthase. The data presented here do support the idea that H2S is

produced by CBS. However, the mechanism for its production is not by the hydrolysis of

cysteine, but rather by a b-replacement reaction utilizing homocysteine and cysteine. This

reaction is essentially identical to the endogenous b-replacement reaction involving

homocysteine and serine, except that cysteine is substituted for serine, resulting in the formation

of H2S instead of H2O. This reaction, like the canonical reaction, is stimulated by addition of

AdoMet. Unlike previous investigators, we did not observed any evidence for the stimulation of

CBS by Ca2+/calmodulin (6).

Although our data is in conflict with some recent work, it is actually quite consistent with

work from the 1960’s examining H2S formation from partially purified serine sulfhydrase

derived from chicken liver. Braunstein and colleagues showed that chicken liver serine

sulfhydrase had a thirty-fold increase in H2S-producing activity when cysteine and homocysteine

were added together, compared to cysteine by itself (22). Later work by this same group showed

that the enzyme identified as chicken liver serine sulfhydrase was in fact enzymatically quite

similar to partially purified rat liver cystathionine beta-synthase, and it was proposed that these

were in fact the same enzyme (23). The data presented here clearly support this interpretation.

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There have been other reports in the literature suggesting that another transsulfuration

enzyme, cystathionine gamma-lyase (CGL) may be important in H2S production in vivo (10,24),

but our data do not support that idea. We did not observe any evidence of significant H2S

formation in yeast extracts using our gel overlay assay when only cysteine was added, even

though yeast has significant levels of CGL activity (24). Furthermore, we failed to observe any

detectable H2S formation in the extracts of mouse livers of animals deleted for endogenous CBS

(KHJ and WDK, unpublished). The evidence that CGL can produce H2S comes from work in

which propargylglycine was shown to inhibit H2S production in crude liver extracts (10). One

possible explanation for this apparent contradiction is that in the context of a crude rat liver

extract the propargylglycine may be affecting other enzymes, resulting in decreased production,

or increased metabolism of H2S.

Our data suggest that the production of H2S from cysteine and homocysteine does occur in

vivo. We found that wild-type human CBS has a Km value for L-cysteine of 6 mM, about three-

fold higher than the Km for serine. We also found that in three substrate reactions (cysteine,

homocysteine, and serine) a small but significant portion of the cystathionine produced came

from cysteine. When the substrates were added at physiological concentrations (as determined in

mouse liver) we found that about 5% of the cystathionine produced came from cysteine. This is

consistent with the observation that H2S levels are at least an order of magnitude less than

cysteine, homocysteine or S-adenosylmethionine levels in the brain levels (25).

Our findings may also have clinical relevance. H2S levels have been shown to be severely

decreased in the brains of Alzheimer’s disease patients and in the aorta of hypertensive rats (25).

If these low levels of H2S are found to be pathogenic in human disease, it may be possible to

increase H2S and lower homocysteine levels by increasing the concentration of cysteine in

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tissues. In fact, it has been shown that pharmacologic doses of N-acetylcysteine can lower

plasma homocysteine levels in humans (26-28). The likely reason for this effect is that N-

acetylcysteine is converted to cysteine inside cells, thereby increasing the concentration of

cysteine and thus driving the conversion of homocysteine to cystathionine. This treatment would

also be expected to increase the production of H2S.

In summary, the findings presented here help clarify the potential role of CBS in the

production of H2S and the reduction of homocysteine, and support the view that CBS may play a

key role in neurobiology.

Acknowledgements—We thank Lisa Henske and Al Knudson for their critical reading of

this manuscript. This work was supported by NIH grants HL57299 and CA06927 and by an

appropriation from the Commonwealth of Pennsylvania. We also acknowledge the work of the

Biotechnology Facility at Fox Chase Cancer Center.

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TABLE I

Cysteine vs. Serine Kinetic parameters

Parameter Serine+Hcy Cysteine+Hcy

Km 1.74 ± 0.23 mM 6.11 ± 2.85 mM

Vmax 10116 ± 329 nmol/mgprotein/min

4353 ± 683 nmol/mgprotein/min

Kcat 10.2/sec 4.39/sec

Kcat/Km 5.9 0.72

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FIGURE LEGENDS

Figure 1. Potential H2S producing reactions catalyzed by CBS. The first reaction shown is the

standard CBS reaction which does not produce H2S. The four alternative reactions are all

capable of producing H2S. The biochemical mechanism by which each reaction proceeds is

indicated.

Figure 2. H2S formation by human CBS. (A) Each panel of the figure shows an identically

loaded Native-Page gel developed under different reaction conditions. The lanes for each panel

are: –, negative control extract (WY218 extract); wt, full length human CBS (1-551); and Tr,

truncated human CBS (1-409). The top three gels were developed in reactions lacking S-

adenosylmethionine (SAM), while the bottom three panels were developed in reactions

containing 0.4 mM SAM. The gels on the left were developed using cysteine + homocysteine

(alternate reaction 1), the middle gels were developed using 2ME+homocysteine (alternate

reaction 2), and the gels on the right were developed only in the presence of cysteine (alternate

reactions 3 and 4). The molecular mass markers in top left are indicated: ferritin (440), catalase

(232), lactate dehydrogenase (140), and bovine serum albumin (67). Markers are shown after

staining with Coomassie blue. (B). 200 ng of purified recombinant CBS was assessed for H2S

production as described in Methods. All reactions were done in triplicate and standard deviation

is as shown. The units for enzyme activity are nmol H2S produced/mg protein/minute. There

was no detectable H2S produced in the reaction containing cysteine alone.

Figure 3. Relative usage of serine and cysteine in cystathionine production. A. Trace of the

440 nm absorbance of an in vitro reaction generated by the BioChrom 30 amino acid analyzer.

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In this reaction, physiologic concentrations of serine (0.72 mM), cysteine (0.47 mM), and

homocysteine (0.52 mM) were combined with 200 ng of purified human CBS. The solid line

shows the reaction incubated on ice for one hour, while the dotted line shows the reaction at

370C. B. Reactions were set up using homocysteine plus serine or cysteine or both at the

concentrations indicated. Reactions were performed as described in Methods. The columns

show the amount of cystathionine formed with the darker texture showing the amount of

cystathionine derived from serine, while the lighter texture shows that derived from cysteine.

The numbers on the bottom row show the ratio of serine vs. cysteine usage in a competitive

reaction using both serine and cysteine with homocysteine.

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Xulin Chen, Kwang-Hwan Jhee and Warren D. Krugercondensation of cysteine and homocysteine

Production of the neuromodulator H2S by cystathionine beta-synthase via the

published online November 1, 2004J. Biol. Chem. 

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