H2S Biogenesis by Human Cystathionine �-Lyase Leads to theNovel Sulfur Metabolites Lanthionine and Homolanthionineand Is Responsive to the Grade of Hyperhomocysteinemia*□S

Received for publication, October 20, 2008, and in revised form, March 3, 2009 Published, JBC Papers in Press, March 4, 2009, DOI 10.1074/jbc.M808026200

Taurai Chiku‡1, Dominique Padovani‡1, Weidong Zhu§1, Sangita Singh‡§, Victor Vitvitsky‡, and Ruma Banerjee‡2

From the ‡Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0606 and the§Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664

Although there is a growing recognition of the significance ofhydrogen sulfide (H2S) as a biological signaling moleculeinvolved in vascular and nervous system functions, its biogene-sis and regulation are poorly understood. It is widely assumedthat desulfhydration of cysteine is the major source of H2S inmammals and is catalyzed by the transsulfuration pathwayenzymes, cystathionine �-synthase and cystathionine �-lyase(CSE). In this study, we demonstrate that the profligacy ofhuman CSE results in a variety of reactions that generate H2Sfrom cysteine and homocysteine. The �-replacement reaction,which condenses two molecules of homocysteine, yields H2Sand a novel biomarker, homolanthionine, which has beenreported in urine of homocystinuric patients, whereas a �-re-placement reaction, which condenses twomolecules of cysteine,generates lanthionine. Kinetic simulations at physiologicallyrelevant concentrations of cysteine and homocysteine, revealthat the �,�-elimination of cysteine accounts for �70% of H2Sgeneration. However, the relative importance of homocysteine-derived H2S increases progressively with the grade of hyperho-mocysteinemia, and under conditions of severely elevated hom-ocysteine (200 �M), the �,�-elimination and �-replacementreactions of homocysteine together are predicted to account for�90% of H2S generation by CSE. Excessive H2S production inhyperhomocysteinemia may contribute to the associated car-diovascular pathology.

H2S is the newestmember of a growing list of gaseous signal-ing molecules that modulate physiological functions (1–3).Concentrations of H2S ranging from 50 to 160 �M have beenreported in the brain (4), where it appears to function as a neu-romodulator by potentiating the activity of the N-methyl-D-aspartate receptor and by altering induction of long termpotentiation in the hippocampus, important for memory andlearning (5). H2S levels in human plasma are reported to be�50�M, and in vitro studies suggest that it functions as a vasodilatorby opening KATP channels in vascular smooth muscle cells (6).

A recent in vivo study has demonstrated the efficacy of H2S inattenuatingmyocardial ischemia-reperfusion injury by protect-ingmitochondrial function (7). The role ofH2S in inflammationis suggested by several studies (8–11); however, the underlyingmechanism is unknown. Remarkably, H2S can also induce astate of suspended animation in mice by decreasing the meta-bolic rate and the core body temperature presumably by inhib-iting cytochrome c oxidase in the respiratory chain (12).

Endogenous H2S is presumed to be generated primarily bydesulfhydration of cysteine catalyzed by the two pyridoxalphosphate (PLP)3-dependent enzymes in the transsulfurationpathway: cystathionine �-synthase (CBS) and cystathionine�-lyase (CSE) (13, 14). In fact, it is widely assumed, based on thereported absences of CSE in the brain (15) and of H2S in thebrain of CBS knock-out mice (16), that CBS is the primarysource of H2S in this organ, whereas CSE plays the equivalentrole in the peripheral vasculature (3). However, recent studieshave demonstrated that CSE is both present and active in thebrain (17, 18) and that H2S is in fact detected in the brains oftransgenic mice lacking CBS (19). The major role of CSE in H2Sbiogenesis in theperipheral systemhas been convincingly demon-strated in CSE knock-out mice, which exhibit significantlyreducedH2S levels in the serumand lowerH2Sproduction rates inaorta and heart (20). The CSE knock-out mice exhibit hyperten-sion and reduced endothelium-dependent vasorelaxation.CSE belongs to the �-family of PLP-dependent enzymes and

catalyzes �,�-elimination of cystathionine to give cysteine,�-ketobutyrate, and ammonia (Fig. 1, reaction 1) (21). In prin-ciple, a variety of CSE-catalyzed reactions leading to H2S for-mation can be considered, including cysteine-dependent�-reactions (Fig. 1, reactions 2, 3, and 6) and homocysteine-de-pendent �-reactions (reactions 4 and 5). An alternative route toH2S synthesis from cysteine catalyzed by CSE has been pro-posed to involve �-elimination of cystine, leading to the inter-mediate formation of thiocysteine (reaction 7), which decom-poses to H2S in a nonenzymatic reaction with other thiols (13,22, 23). However, the significance of cystine as a source of H2S,in the reducing intracellular environment is uncertain.In this study, we have elucidated the kinetics of H2S biosyn-

thesis from cysteine and homocysteine catalyzed by recombi-nant human CSE. The kinetic data have been utilized to simu-

* This work was supported, in whole or in part, by National Institutes of HealthGrant HL58984 (to R. B.). This work was also supported by an AmericanHeart Association postdoctoral fellowship award (to W. Z.).

□S The on-line version of this article (available at http://www.jbc.org) con-tains Tables S1–S3.

1 These authors contributed equally to this work.2 To whom correspondence should be addressed: 3320B MSRB III, 1150 W.

Medical Center Dr., University of Michigan, Ann Arbor, MI 48109-0606. Tel.:734-615-5238; E-mail: rbanerje@umich.edu.

3 The abbreviations used are: PLP, pyridoxal 5�-phosphate; CBS, cystathionine�-synthase; CSE, cystathionine �-lyase; DTNB, dithiobisnitrobenzene;HPLC, high pressure liquid chromatography; MS, mass spectrometry; HCys,homocysteine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 17, pp. 11601–11612, April 24, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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late the rate of H2S production by CSE at physiologicallyrelevant concentrations of substrates and at three concentra-tions of homocysteine, to mimic normal, mild, and severehyperhomocysteinemia, and the simulated data have been val-idated experimentally. The simulations predict that the relativecontribution of homocysteine versus cysteine toH2S biogenesisby CSE increases with the grade of hyperhomocysteinemia.Our studies have led to the identification of two novel sulfurmetabolites generated as byproducts of H2S synthesis byCSE, lanthionine and homolanthionine. The latter couldserve as a biomarker for H2S production under hyperhomo-cysteinemic conditions.

EXPERIMENTAL PROCEDURES

Purification of Human CSE

Recombinant human CSE (polymorphic variant S403) wasexpressed in the Escherichia coli strain BL21(DE3) using anexpression plasmid generously provided by Dr. Marcus Wahl(Max Planck Institute, Mantinsried, Germany). The protein waspurified as described previously (24) with the following modifica-tion. After the Superdex S-200 (Sigma) size exclusion column, theactive fractions were pooled, concentrated, and dialyzed against100 mMHepes buffer, pH 7.4, before being stored at �80 °C. Theconcentration of CSE was determined using the Bradford reagent(Bio-Rad) with bovine serum albumin as a standard.

Enzyme Activity Assays

The following assayswere employed to assessCSE activity. Inall assays, the concentration of the variable substrate ranged

from 0.2 � Km1 to 30 � Km1. Oneunit of activity is defined as theamount of enzyme needed to form 1�mol of product min�1.Detection of Cysteine—The DTNB

assay was used to measure cysteineproduced by CSE from cystathi-onine, as described previously (25).Briefly, 970 �l of Hepes buffer (100mM, pH 7.4) containing variousamounts of a diasteromeric mix ofcystathionine was mixed with 10 �lof 0.1 M DTNB (in ethanol) andincubated at 37 °C for 3 min. PLPwas omitted from the reaction mix-ture, since its addition consistentlyleads to a slight inhibition of theenzymatic activity. Enzyme (20�l of1 mg/ml protein) was added to ini-tiate the reaction, and an increase inabsorption at 412 nm due to forma-tion of the nitrobenzene thiolateanion was monitored for 1 min in aCary100 UV-visible spectropho-tometer thermostatted at 37 °C.Control experiments lacking CSE orsubstrate yielded the backgroundrates for the reaction of DTNB withthe free thiols of CSE or the impuri-

ties contained in cystathionine (�90% purity) and were sub-tracted from the enzyme assay data. A molar extinction coeffi-cient of 13,600 M�1 cm�1 was used to estimate theconcentration of cysteine generated.Detection of H2S—H2S generation was measured in one of

two ways. For in-gel assays, H2S production was assayed byreaction with lead acetate using a modification of a previouslydescribed method (26, 27). Purified CSE (40 �g/lane) wasloaded into wells of a native 4–15% gradient Tris-glycine gel(Bio-Rad). Immediately after gel electrophoresis (at 4 °C), thegel was cut between the lanes, and the strips were soaked for 6 hat room temperature in 40 ml of the reaction mixture (100 mM

Hepes buffer (pH7.4), 0.4mM lead acetate, and substrates: reac-tion 1 (30mML-homocysteine), reaction 2 (10mML-cysteine, 30mM L-homocysteine), or reaction 3 (10 mM L-cysteine)). BandsproducingH2S developed a dark brown color that was analyzedusing the Gel Doc 2000 gel documentation system (Bio-Rad).Production of H2S by CSE from different substrates was meas-

ured in a spectrophotometric assay in which the reaction of H2Swith lead acetate to form lead sulfidewasmonitored continuouslyby the increase inabsorptionat 390nm.After the reactionmixture(980 �l) containing 100 mM Hepes buffer (pH 7.4), 0.4 mM leadacetate, and varying concentrations of substrate (homocysteine,cysteine, or both) was preincubated at 37 °C for 4 min, 20 �g ofCSEwas added to the assaymixture to initiate the reaction, whichwas monitored at 37 °C for 3 min. Lead acetate (0.4 mM) did notinhibit CSE, as determined in the DTNB assay described above.The molar extinction coefficient for lead sulfide under these con-

FIGURE 1. Cystathionine cleavage and H2S-generating reactions catalyzed by CSE.

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ditions was determined to be 5,500 M�1 cm�1 using NaHS as astandard.Detection of �-Ketoacid Products—Determination of �-keto-

acids generated in the CSE assay was performed as described (28,29). Briefly, 1 ml of the assay mixture containing 100 mM Hepesbuffer (pH 7.4) and varying concentrations of homocysteine orcysteinewas preincubated for 5min at 37 °C, and the reactionwasinitiated by adding 20–50 �g of CSE. At the desired time points,200-�l aliquots of the reaction mixture were quenched by adding200�l of 10% trichloroacetic acid. The precipitated proteins wereremoved by centrifugation at 14,000� g for 10min, and 200�l ofthe supernatant was mixed with 500 �l of 0.5 M sodium acetatebuffer, pH 5.0, and 200 �l of 0.1% 3-methyl-2-benzothiazolinonehydrazone hydrochloride and then incubated at 50 °C for at least30 min. The control experiment lacking substrate was performedin parallel. After the mixture cooled down to room temperature,the absorbance at 316 nmwas read. The concentration of�-keto-butyrate in the reaction mixture was calculated using a standardcurve generated with known concentrations of �-ketobutyrate.The concentration of pyruvate in the reactionmixture was calcu-lated similarly by measuring the absorbance at 324 nm and usingthe appropriate standard curve.HPLC Analysis of Cystathionine—The HPLC method was

used to estimate the rate of cystathionine formation (reaction6) at 10 mM cysteine and varying concentrations of homo-cysteine. The concentration of cystathionine was determinedfollowing o-phthaldialdehyde derivatization, essentially asdescribed previously (30, 31). Briefly, the enzymatic reactionwas stopped by the addition of an equal volume of 10% trichlo-roacetic acid, and the precipitated protein was removed by cen-trifugation. The supernatant was neutralized to pH 7–8 with asmall amount of saturated K2CO3 and diluted 1:4 with boratebuffer (0.2 M, pH 9.6). A 50-�l aliquot of each sample wasremoved and derivatizedwith 25�l of o-phthaldialdehyde solu-tion (15 mM o-phthaldialdehyde, 30 mM 2-mercaptoethanol,and 10% methanol in 0.2 M sodium borate buffer, pH 9.6) in anautosampler (Agilent 1100 series) for 1 min at 10 °C. A 10-�laliquot of the derivatized sample was then injected into theHPLC column (ZORBAX Eclipse XDB-C18 (5-�m) analyticalcolumn 4.6 � 150 mm) and eluted at a flow rate of 1 ml/minwith buffers A (80% 0.1 M sodium acetate and 20% methanol,pH 4.75) and B (20% 0.1 M sodium acetate and 80% methanol,pH4.75). The following increasing gradient of buffer Bwas usedfor elution: 0–10 min, 30–60%; 10–15 min, 60–100%; 15–20min, 100%; 20–22 min, 100–30%; 22–30 min, 30%, with a cor-responding decrease in the percentage of bufferA. The detectorwas set at 340-nm excitation and 450-nm emission wave-lengths. Under these conditions, cystathionine eluted with aretention time of 14.06 min. The concentration of cystathi-onine was determined using calibration coefficients obtainedwith the standard. The sameHPLCmethodwas used to analyzewhether serine and homoserine were produced by the CSE-catalyzed �,�-elimination (reaction 2) and �,�-elimination(reaction 4) reactions, respectively. The retention times forthese compounds were 4.17 min (serine) and 5.26 min forhomoserine.

H2S Production at Physiologically Relevant Concentrations ofSubstrate—H2S formation was detected using the lead acetateassay described above with the following exception. The reac-tion mixture (1-ml final volume) contained 100 mM Hepesbuffer (pH7.4), 0.4mM lead acetate, 5�Mcystathionine, 100�Mcysteine, and either 10, 40, or 200 �M homocysteine. Followingincubation at 37 °C for 4 min, the reaction was initiated by theaddition of 100 �g of CSE (corresponding to 2.2 �M activesites), and the reaction wasmonitored at 390 nm for 3min. Thehigher concentration of protein was necessary for monitoringthe slow reaction rates at these low substrate concentrations.We note that the total substrate concentration for H2S genera-tion (i.e. cysteine and homocysteine) varied from 50- to 140-fold excess over the concentration of active sites, and �2–10turnovers were completed during the 3-min time course of theassay.

Mass Spectrometric (MS) Analysis of Reaction Products

For the qualitative analysis of other products in H2S genera-tion reactions, a Q TRAPTM mass spectrometer (Applied Bio-systems) equipped with a Turbo ion spray source operated inthe positive ionmode was employed. Data acquisition was con-ducted using Analysis software (Applied Biosystems) with abuilt-in information-dependent acquisition scan function. Thesupernatant from the assay mixture obtained after protein pre-cipitation by trichloroacetic acid was injected into the massspectrometer. Control reaction mixtures from which CSE wasomitted were run separately.

Analysis of Kinetic Data

Cystathionine, the substrate for CSE, is a condensation prod-uct of two amino acids, serine and homocysteine. The activesite pocket therefore has binding determinants for two aminoacids. In the H2S-generating reactions catalyzed by CSE (reac-tions 2–6), either one (reaction 2 and 4) or both (reactions 3, 5,and6) amino acid binding pockets are occupied.We refer to thekinetic parameters associated with the single substrate reaction(i.e. ignoring H2O) as Km1 and Vmax1. The parameters Km2 andVmax2 then refer to substrate binding at the second site and thereaction velocity of the bimolecular reaction involving twoamino acids, respectively.Cysteine Production from Cystathionine—The Km and Vmax

values for reaction 1were determined directly fromMichaelis-Menten kinetic analysis using the DTNB assay described aboveand Equation 1.

�cysteine �Vmax�CST�

Km�CST� � �CST�(Eq. 1)

We note that commercially available cystathionine is a mix-ture of diastereomers of which only one, the L,L-isomer, isexpected to serve as substrate for CSE. Hence, the substrateconcentrationwas divided by a factor of 4 to obtain the value forKm that is reported.Pyruvate or �-Ketobutyrate Generation—The Km and Vmax

values for CSE-catalyzed pyruvate (reaction 2) or �-ketobu-tyrate (reaction 4) production from cysteine or homocysteine,

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respectively, were determined using the �-ketoacid assaydescribed above and Equations 2 and 3.

�pyruvate �Vmax�Cys�

Km1�Cys� � �Cys��1 ��Cys�

Ki� (Eq. 2)

��-ketobutyrate �Vmax�HCys�

Km1�HCys� � �HCys��1 ��HCys�

Ki� (Eq. 3)

To account for the observed substrate inhibition, an inhibitionconstant (i.e. the Ki term) was included in Equations 2 and 3.H2SProduction fromCysteine orHomocysteine—Inprinciple,

the reactions for H2S production by CSE can follow either abinary (ping-pong) or ternary or sequential (random orordered) mechanism. Hence, the experimental data for H2Sproduction from cysteine (i.e. reactions 2 3) or from homo-cysteine (i.e. reactions 4 5) monitored by the continuous leadacetate assay described above were fitted using Equations 4–6.

�H2S �Vmax1�Cys�

Km1�Cys� � �Cys��

1

1 ��Cys�

Km2�Cys�

�Vmax2

1 �Km1�Cys�

�Cys��

Km2�Cys�n

�Cys�n �1 �Km1�Cys�

�Cys� �(Eq. 4)

�H2S �Vmax1�Cys�

Km1�Cys� � �Cys��

1

1 ��Cys�

Km2�Cys�

�Vmax2

1 �Km1�Cys�

�Cys��

Km2�Cys�n

�Cys�n �1 �Kd1�Cys�

�Cys� �(Eq. 5)

�H2S �Vmax1�Cys�

Km1�Cys� � �Cys��

1

1 ��Cys�

Km2�Cys�

�Vmax2�Cys��Cys�n

�Cys��Cys�n � Km1�Cys��Cys�n � �Cys�Km2�Cys�n (Eq. 6)

Binding of the secondmole of cysteine or homocysteine in reac-tions 3 and 5, respectively, will affect the Vmax1 values for H2Sformation in the unimolecular reactions2 and4. To account forthis, an inhibition term where Ki Km2 was introduced asshown in Equations 4–6.Equations 4–6 describe a random sequential, ordered

sequential, and ping-pong mechanism, respectively. A Hillcoefficientwas included in these equations to account for coop-erativity of binding for the second substrate, which was indi-cated by the kinetic data and our fitting attempts. Equations4–6, as written, describe vH2S from cysteine (i.e. reactions 2 3) for the alternative mechanisms.For analysis of vH2S from homocysteine (i.e. reactions 4 5),

Equations 4–6 were also employed, making the corresponding

substitutions (i.e. [Cys] for [HCys], etc.). Unlike cysteine, thedependence of the reaction velocity for H2S generation onhomocysteine concentration did not show two well separatedphases. Hence, the values for Km1(Hcys), Vmax1, and Ki for �-ke-tobutyrate generation (obtained from Equation 3) were used asinput parameters in Equations 4–6. The quality of fits obtainedfor the ordered sequential mechanism was significantly worsethan for the other two mechanisms (Tables S1 and S2). In con-trast, the quality of fits for the ping-pong versus the randomsequential mechanism was indistinguishable.Cystathionine Production from Cysteine Plus Homocysteine—

The Km and Vmax values for reaction 6 were determined usingthe HPLC assay for cystathionine formation, as describedabove, and Equation 7.

�CST �Vmax2�Cys��HCys�n

�Cys��HCys�n � Km1�Cys��1 ��HCys�

Km1�HCys���HCys�n � �Cys�Km2�HCys�

n�1 ��Cys�

Km2�Cys��

(Eq. 7)

In principle, reaction 6 can follow either a binary or ternarymechanism with either cysteine or homocysteine binding first.However, a reasonable fit was only obtained for the ping-pongmechanism where cysteine binds first (Table S3). Equation 7describes a ping-pong mechanism, in which competitive inhi-bition terms for the binding of the first and second substrateswere included, since the simultaneous presence of both sub-strates leads to competition at each binding site by the othersubstrate (Ki(Hcys) Km1(HCys) and Ki(Cys) Km2(Cys)).H2S Production from Cysteine and Homocysteine—Next, the

goodness of the kinetic parameters obtained for reactions 2–6was assessed by fitting the experimental data for H2S formationobtained in the presence of 10mMcysteine and varying concen-trations of homocysteine. In this set of experiments, theobserved rate of H2S production represents the sum of reac-tions 2–6, as described by Equation 8.

�H2S � �2 � �3 � �4 � �5 � �6 (Eq. 8)

The values of v2–v6 corresponding to the reaction velocities for2–6 were computed using Equations 9–13 for the ping-pongmechanism,

�2 �Vmax1�Cys�

Km1�Cys��1 ��HCys�

Km1�HCys��� �Cys�

�1

1 ��Cys�

Km2�Cys��

�HCys�

Km2�HCys�

(Eq. 9)

�3 �Vmax2�Cys��Cys�n

�Cys��Cys�n � Km1�Cys��1 ��HCys�

Km1�HCys���Cys�n � �Cys�Km2�Cys�

n�1 ��HCys�

Km2�HCys��

(Eq. 10)

�4 �Vmax1�HCys�

Km1�HCys��1 ��Cys�

Km1�Cys��� �HCys�

�1

1 ��HCys�

Km2�HCys�(Eq. 11)

�5 �Vmax2�HCys��HCys�h

�HCys��HCys�h � Km1�HCys��1 ��Cys�

Km1�Cys���HCys�h � �HCys�Km2�HCys�

h�1 ��Cys�

Km2�Cys��

(Eq. 12)

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�6 �Vmax2�Cys��HCys�k

�Cys��HCys�k � Km1�Cys��1 ��HCys�

Km1�HCys���HCys�k � �Cys�Km2�HCys�

k�1 ��Cys�

Km2�Cys��

(Eq. 13)

where n, h, and k represent theHill coefficient for the binding ofthe second substrate in reactions 3, 5, and 6, respectively, and

the kinetic parameters were ob-tained as described above. A termfor competitive inhibition (Km(1 [I]/Ki)) was introduced in Equations9–13, since the simultaneous pres-ence of both substrates leads tocompetition for each binding site bythe other substrate.Determination of the Rates of H2S

Production at Physiological Sub-strate Concentrations—The contri-butions of the various CSE-cata-lyzed reactions to total H2Sproduction were computed at nor-mal, medium, and high homocys-teine concentrations (10, 40, and200 �M, respectively), using Equa-tions 9–13. The following steadyconcentrations of substrates wereused: [homocysteine] 10, 40, or200 �M; [cysteine] 100 �M; [cys-tathionine] 5 �M. The Ki for cys-tathionine was ignored, since theconcentration of cystathionine usedto simulate physiological conditionsis low (5�M), whereas theKi for cys-tathionine forH2S production is rel-

atively high (0.78 � 0.1 mM). For instance, the inclusion of theKi term for cystathionine affected the value for v2 by �2%. Thevalues for v1 corresponding to reaction 1, at varying homocys-teine concentrations, were computed using Equation 14,

�1 �Vmax�CST�

Km�CST��1 ��Cys�

Km1�Cys��

�HCys�

Km1�HCys�� � �CST�

(Eq. 14)

where cysteine and homocysteine act as competitive inhibitorsfor binding of cystathionine. Ki(Cys) and Ki(HCys) wereassumed to be equal to Km1(Cys) and Km1(HCys) respectively.

The resulting reaction rates (v1–v6) for reactions 1–6 werethen used to calculate the turnover numbers (i.e. v/[E]) for eachreaction at the substrate concentrations described above andare expressed per mole of CSE active site.

RESULTS

Purification and Biophysical Characterization of CSE—Purifi-cation of recombinant human CSE was accomplished in threechromatographic steps, and the purified protein was judged to be�95% pure by gel electrophoresis (Fig. 2, inset). The typical yieldwas�20mgofpureprotein/liter of culture.The specific activityofas-purified CSE is 3.1 � 0.1 units/mg in the DTNB assay withcystathionine as substrate and is similar to the value publishedpreviously (2.5 units/mg) (25). As expected, the absorption spec-trum of purified CSE is typical of a PLP-dependent enzymewith amaximum at 428 nm and a 280:428 nm ratio of �1:6 (Fig. 2).H2S Production by Human CSE—The ability of human CSE to

generate H2S was first assessed by an in-gel activity assay. For thisexperiment, native gel strips containing equal amounts of purified

FIGURE 2. Purification and characterization of recombinant human CSE. The UV-visible absorption spec-trum of CSE (1.7 mg/ml in 70 mM Tris-HCl buffer, pH 8.1, containing 2 mM EDTA and 150 mM NaCl) exhibits amaximum at 428 nm due to PLP. Inset, purity of CSE (20 �g) detected on a 12% SDS-polyacrylamide gel byCoomassie Blue staining. The sizes of the molecular weight standards (M) are shown.

FIGURE 3. H2S generation by CSE. The reaction of H2S with lead acetate toform lead sulfide was monitored by the increase in absorbance at 390 nmunder quasi-steady-state conditions, as described under “Experimental Pro-cedures,” using as substrates 30 mM homocysteine (a), 30 mM homocysteineplus 10 mM cysteine (b), and 10 mM cysteine (c). Inset, in-gel activity staining ofCSE. 40 �g of CSE was loaded in each lane and separated on a 4 –15% nativegradient gel, and H2S-producing activity was detected as described under“Experimental Procedures.” Although the major band corresponds to the nativetetrameric form of CSE, a small proportion appear as high order oligomers. Themolecular weight markers (M) were stained with Coomassie Blue.

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CSE were exposed to the following conditions (30 mM homocys-teine, 30mMhomocysteineplus 10mML-cysteine, or 10mML-cys-teine). H2S generation was revealed by the appearance of a darklead sulfide-containing band on the gel. As shown in Fig. 3 (inset),themost intense bandswere seen in thepresenceof homocysteine(lane a). When both homocysteine and cysteine were present inthe reactionmixture, lowerH2Sproductionwas observed (lane b),whereas cysteine alone supported the lowest level of H2S genera-tion (lane c). These results indicate that at saturating concentra-tions, homocysteine rather than cysteine is themore effective sub-strate for H2S generation by CSE. We note that CSE migrates asthree bands on the native gel, indicating that although the majorpopulation is a tetramer (fastestmigrating band), aminor propor-tion exists as higher order oligomers.The kinetics of H2S generation by CSE were further charac-

terized using a continuous spectrophotometric assay. The spe-cific activities under Vmax conditions for H2S formation are6.6 � 0.5 units/mg from homocysteine and 1.2 � 0.3 units/mgfrom cysteine (Table 1). As also seen in Fig. 3, the rate of H2Sformation from homocysteine is higher than from cysteine orfrom homocysteine plus cysteine. The decrease in the initialvelocity of H2S formation when both substrates are present incomparison with the rate observed with homocysteine aloneresults from the occupancy of a portion of the enzyme activesites by the slower substrate, cysteine. This has the net effect ofdecreased total H2S flux generation. Conversely, the apparentactivation of H2S production when both substrates are presentin comparison with cysteine alone results from the fraction ofthe enzyme that is catalyzing homocysteine-dependent H2Sproduction, which occurs at a faster rate than from cysteine.Propargylglycine, a suicide inhibitor of CSE (32), completelyblocked H2S formation (not shown). Unlike rat CSE thatreportedly uses cystine (Fig. 1, reaction 7) rather than cysteineto generate H2S (23), H2S formation from cystine was notobserved with human CSE (data not shown).Product Analysis of H2S-producing Reactions—To distin-

guish between the multiple routes for H2S generation by CSE(Fig. 1, reactions 2–6), reaction products were analyzed bymass spectrometry, HPLC, and UV-visible absorption spec-troscopy for detection of the keto acids, pyruvate and �-keto-butyrate (28). In the presence of cysteine, pyruvate and a novelmetabolite, lanthionine (m/z 209; Fig. 4), were observed, con-sistentwith an�,�-elimination reaction (reaction2) and a�-re-placement reaction (reaction 3). In the presence of homocys-

FIGURE 4. Product analysis by MS of the CSE-catalyzed reactions in thepresence of homocysteine plus cysteine (A), homocysteine alone (B), orcysteine alone (C). Parent ions with m/z values of 122 (cysteine), 136 (hom-ocysteine), 209 (lanthionine), 223 (cystathionine), 237 (homolanthionine),241 (cystine), and 269 (homocystine) are seen.

TABLE 1Kinetic parameters for reactions catalyzed by CSEAll values are the average of at least three independent experiments � S.D.

Reaction Number Vmax Km Ki n kcat kcat/Km

unitsa/mg mM mM s�1 mM�1 s�1

�-Elimination of cystathionine 1 3.1 � 0.1 0.28 � 0.03 2.3 8.2Pyruvate generation 2 0.42 � 0.07 3.7 � 1.1 32.0 � 9.5 0.31 0.08H2S generation from Cys 2 0.6 � 0.1 1.7 � 0.7 33 � 8 0.47 0.27

3 1.2 � 0.3 33 � 8b 3.0 � 1.0 0.85 0.026c�-Ketobutyrate generation 4 1.2 � 0.3 2.7 � 1.4 14.5 � 6.8 0.92 0.35H2S generation from HCys 4 1.2 � 0.3 2.7 � 0.85 14.5 � 6.8 0.92 0.35

5 6.6 � 0.47 5.9 � 1.2b 1.8 � 0.6 4.9 0.83cCystathionine generation 6 0.20 � 0.03 12.0 � 5.4d 1.6 � 0.7 0.15 0.012d

a A unit of activity is defined as 1 �mol of product generated min�1 at 37 °C.b The Km value corresponds to Km2 (i.e. for binding of substrate to site 2).c The kcat/Km value is reported relative to Km2.d The value represents Km(HCys), and the kcat/Km value is reported relative to Km(HCys).

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teine and cysteine, cystathionine (m/z 223; Fig. 4) wasdetected, consistentwith a�-replacement reaction (reaction6).In the presence of homocysteine alone, not only was �-ketobu-tyrate detected, consistentwith an�,�-elimination (reaction4),but a newmetabolite, homolanthionine (m/z 237; Fig. 4), wasseen, indicating the �-replacement of one molecule of homo-cysteine by another (reaction 5). Serine and homoserine, theproducts of �- and �-elimination reactions, respectively (reac-tions 2 and 4), were not detected by HPLC, but their down-stream products, pyruvate and �-ketobutyrate, respectively,were observed. Lanthionine and homolanthionine are struc-tural homologs of cystathionine that differ by the absence orpresence of an extra methylene group, respectively. The iden-tity of homolanthionine was confirmed by MS/MS analysis inwhich two daughter ion peaks were assigned with m/z 102(corresponding to HOOCCH(NH2)CH2CH2)) and 134 (corre-sponding to SCH2CH2CH(NH2)COOH) that were 14 atomicmass units heavier than the corresponding peaks seen inthe MS/MS spectrum of cystathionine (not shown). The

identity of lanthionine was con-firmed by MS/MS analysis, whichwas identical to that of a commer-cial sample of lanthionine in whichtwo daughter ion peaks wereassigned with m/z 120 (corre-sponding to SCH2CH(NH2)COOH)and m/z 192 (correspondingto loss of NH3 from lanthionine)(not shown). These data establishthat homolanthionine and lanthi-onine produced by CSE arederived from homocysteine andcysteine, respectively.Effect of Nitric Oxide (NO) on

H2S-producing Activity of CSE—Previously, it has been reported thatthe NO donor, sodium nitroprus-side, increases the endogenous lev-els ofH2S in vascular tissues (6). Themechanism of this increase wasproposed to involve either an NO-induced increase in CSE activity orNO-dependent up-regulation ofCSE expression (6). However, weobserved no effect of sodium nitro-prusside on H2S production by CSE(data not shown), indicating that theeffect ofNO is not at the level ofCSEactivity.Kinetics of H2S Generation by

CSE—Product analyses provideddirect evidence for five of the sixpossible CSE-dependent H2S-gen-erating reactions described in Fig.1 (i.e. reactions 2–6). The kineticsof pyruvate (reaction 2) and �-ke-tobutyrate (reaction 4) formationfrom cysteine and homocysteine,

respectively, and the kinetics of H2S formation from thesame substrates (i.e. reactions 2 3 or reactions 4 5) areshown in Fig. 5. The kinetic data were fitted to alternativemechanisms (i.e. binary versus ternary), and the data are pre-sented in Tables S1 and S2. The values of the kinetic param-eters obtained from fits to the ping-pong mechanismallowed deconvolution of the Km and Vmax values associatedwith each of the four reactions (Table 1). The dependence ofthe rate of H2S formation on cysteine concentration is mark-edly biphasic (Fig. 5B). CSE exhibits a considerably higheraffinity for cysteine binding to site 1 (3.7 � 1.1 mM) than tosite 2 (33 � 8 mM), and cooperativity for binding of thesecond mole of cysteine was seen (n 3 � 1).Deconvolution of the two phases contributing to the rate of

H2S formation fromhomocysteine (Fig. 5D) reveals that theKmfor site 1 is 2-fold lower than for site 2 (2.7 � 1.4 and 5.9 � 1.2mM, respectively). The kinetics of reaction 6 (i.e. the condensa-tion of homocysteine and cysteine) were monitored by the rateof cystathionine formation. The kinetic data could only be fit

FIGURE 5. Kinetics of �-ketoacids and H2S generation by CSE in the presence of cysteine or homocys-teine. Shown are the kinetics of pyruvate (A) (reaction 2) or �-ketobutyrate (C) (reaction 4) generation fromcysteine or homocysteine, respectively. Also shown are kinetics of H2S generation from cysteine (B) (reactions2 3) or from homocysteine (D) (reactions 4 5). The contributions of the component reactions (v2 and v3 inB and v4 and v5 in D) to the net rate of H2S generation are shown. Each data point represents the mean � S.D.of at least three independent experiments. The data were analyzed as described under “Experimental Proce-dures,” and the kinetic parameters obtained from these plots are shown in Table 1.

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with a ping-pong mechanism in which cysteine is the first sub-strate to bind (Table S3). The relative catalytic efficiencies (i.e.kcat/Km) for the five H2S-generating reactions follow the order5 4 2 3 6 (Table 1).

The kinetic parameters obtained for reactions2–6were thenemployed to simulate the kinetics of H2S formation in the pres-ence of cysteine and homocysteine (Fig. 6A). The excellent cor-respondence between the simulated and experimental datasupports the validity of the kinetic parameters reported inTable 1.Relative Contributions of the CSE-catalyzed Reactions to H2S

Generation—Since the cleavage of cystathionine (reaction 1)represents the primary function of CSE in the transsulfurationpathway, it is pertinent to compare the catalytic efficiency of

this reaction with those of the sidereactions leading to H2S generation(Table 1). Under Vmax conditions,the most efficient H2S-generatingreaction (i.e.�-replacement of hom-ocysteine (reaction 5)) exhibits akcat/Km value that is �10-fold lowerthan that for the �,�-elimination ofcystathionine. Furthermore, the Kmfor cystathionine (0.28 � 0.03 mM)is significantly lower than forhomocysteine.In the cell, the substrate concen-

trations are low compared withtheir Km values (i.e. [S] �� Km).Under these conditions, most of theenzyme active sites are unoccupied,and the partitioning of CSE into thevarious H2S-generating reactions isgoverned by the rate of each reac-tion (i.e. v Vmax[S]/Km). This isdistinct from the situation under invitro steady-state assays conductedat high concentrations of substrate,where the kcat/Km ratio determinesthe enzyme specificity for compet-ing substrates. Thus, in the cell, sub-strate availability will play a crucialrole in determining the partitioningof CSE between competing reactionpaths, and regulatory mechanismsare likely to exist that lead toenhanced or diminished productionof H2S and to the diversion of CSEfrom its role in the transsulfurationpathway.Using the kinetic parameters

described in Table 1, we simulatedthe relative contributions of each ofthe reactions to total H2S produc-tion at three concentrations of hom-ocysteine, representing normal (10�M) versus moderate (40 �M) andsevere (200 �M) hyperhomocys-

teinemia (Tables 2 and 3 and Fig. 6, B and C). According to oursimulations, under normal conditions, �,�-elimination of cys-teine (reaction 2) is predicted to be the major source of CSE-derived H2S, accounting for �70% of the total (Table 3 and Fig.6,B andC). The�,�-elimination of homocysteine (reaction4) isthe next significant contributor (�29%), whereas the �- and�-replacement reactions (reactions 3, 5, and 6) are of negligibleimportance. The balance between the reaction shifts, however,with increasing concentrations of homocysteine such that the�,�-elimination of homocysteine (reaction 4) becomes a signif-icant source of H2S atmoderate and the principal source of H2Sat severely elevated homocysteine concentrations (Fig. 6, B andC). The condensation reaction between 2mol of cysteine (reac-tion 3) is aminor contributor to the net H2S pool. Since the rate

FIGURE 6. The relative contributions of reactions 2– 6 to H2S production at varying homocysteine con-centrations. A, the rate of H2S production (E) observed in the presence of 10 mM cysteine and varying con-centration of homocysteine in 0.1 M Hepes buffer, pH 7.4, at 37 °C. Each data point represents the mean � S.D.of three independent experiments. The relative contributions of the individual reactions (v2–v6) to the net rateof H2S production (solid line) were simulated using the kinetic parameters reported in Table 1 and as describedunder “Experimental Procedures.” B, the contributions of the individual reactions (2– 6) to H2S production byCSE were calculated at normal (10 �M), moderate (40 �M), and high (200 �M) concentrations of homocysteineand physiological concentrations of cystathionine and cysteine (5 and 100 �M, respectively) (Table 2). Thereaction numbers are indicated above the bar graphs on the left. C, the relative proportions of CSE-derived H2Sfrom cysteine versus homocysteine at three concentrations of homocysteine and physiological concentrationsof cysteine and cystathionine (Table 3).

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of reaction 5 has a square dependence on the concentration ofhomocysteine, it exhibits the greatest sensitivity to increasinghomocysteine concentrations, changing�230-fold between 10and 200�Mhomocysteine (Table 3).Homolanthionine produc-tion could therefore be a useful biomarker for H2S productionat high homocysteine concentrations. Generation ofH2S by the�-replacement of homocysteine (reaction 5) accounts for�13%) of total H2S generation by CSE under conditions ofsevere hyperhomocysteinemia. Cystathionine formation (reac-tion 6) is also predicted to rise with increasing homocysteine(Table 3), but it is unlikely to build up, since it is an efficientsubstrate for CSE. Under conditions of cystinuria, lanthionineproduction via reaction 3 would be expected to increase.Comparison of Experimental versus Simulated Kinetic Data

at Physiological Concentrations of Substrates—To test thevalidity of the simulations described above, the kinetics of theCSE-catalyzed production of H2S at substrate concentrationschosen to mimic their physiological levels were determined. Atlow homocysteine concentrations (10 �M), 70% of H2S is pre-dicted to result from the �,�-elimination of cysteine (v/[E] 0.0081 s�1) and 29% from the �,�-elimination of homocysteine(v/[E] 0.00335 s�1) (Table 2). The experimentally observedturnover number for H2S formation under these conditionswas 0.012 � 0.001 s�1 and similar to the calculated value of0.0115 s�1. As the concentration of homocysteine increases,the net rate of H2S production is expected to increase. In addi-tion, the proportion of H2S that is derived from homocysteineincreases from 29 to 63 to 90% as homocysteine increases from10 to 40 to 200�M, respectively (Table 3). In contrast, the rate ofH2S production from cysteine is virtually unchanged, whereasthe proportion of cysteine-derived H2S decreases from 70 to 37to 10%. The value of v/[E] for H2S generation is predicted to

increase to 0.0217 and 0.0781 s�1 at 40 and 200 �M homocys-teine, respectively. The experimentally observed v/[E] valuesfor H2S formation under these conditions were found to be0.015 � 0.007 s�1 (40 �M homocysteine) and 0.06 � 0.001 s�1

(200 �M homocysteine). The excellent correspondencebetween the experimentally measured and predicted turnovernumbers for H2S production support the validity of the simu-lations reported here.

DISCUSSION

The nonenzymatic liberation of H2S from organic polysul-fides in garlic bulbs has been reported recently and provides amechanistic explanation for the vasoactivity of dietary garlic(33). However, despite the growing interest in H2S biology andthe therapeutic potential of H2S-releasing compounds (2), sur-prisingly little is known about the enzymatic production of thisgas and how it may be influenced by changes in sulfur aminoacid levels in disease states.Since the enzymes in the transsulfuration pathway, CBS and

CSE, catalyze elimination/addition reactions at the �- and�-positions of sulfur-containing amino acids, respectively, theyare logical candidates for the generation of H2S. However, con-flicting reports in the literature ascribe the generation ofH2S byCBS and CSE to different substrates. For example, cystine wasproposed to be the preferred substrate for H2S by CSE (23),whereas the �-replacement of cysteine by homocysteine isreported to be the preferred route for H2S generation by CBS(27). In this study, we have investigated the various reactionscatalyzed byCSE that result inH2S biogenesis and, as side prod-ucts, the novel amino acids, lanthionine and homolanthionine.The multitude of H2S-generating reactions (Scheme 1) and therelatively high Kms for homocysteine and cysteine exhibited byCSE versus the intracellular concentrations of these aminoacidsmakes kinetic analysis complex and necessitates the use ofsimulations to deconvolute the contributions of different sub-strates to the overall H2S pool.

The reaction catalyzed by CSE in the transsulfuration path-way involves elimination at the �-carbon of cystathionine. Wefind that the catalytic efficiency (kcat/Km) of the canonical cys-teine elimination reaction fromcystathionine is�20- and�30-fold higher than for H2S elimination from homocysteine andcysteine, respectively (Table 1). At physiologically relevantconcentrations of homocysteine (10 �M), cysteine (100 �M),and cystathionine (5 �M), the turnover number for cystathi-onine cleavage (0.039 s�1) is still 5-fold greater than for cysteine

TABLE 2Kinetic parameters for the CSE-catalyzed reactions at varying homocysteine concentrations

Substrate Reaction numbera Vmaxb Km1 (Km2)c v/�E�, 10 �M HCysd v/�E�, 40 �M HCys Changee v/�E�, 200 �M HCys Change

units/mg mM s�1 sec�1 -fold s�1 -foldCystathionine 1 3.10 0.28 0.03916 0.03874 0.99 0.03666 0.94Cys 2 0.42 3.7 8.1 � 10�3 8.03 � 10�3 0.99 7.5 � 10�3 0.92Cys Cys 3 1.2 3.7 (33) 2.85 � 10�8 2.83 � 10�8 0.99 2.76 � 10�8 0.97HCys 4 1.2 2.7 3.35 � 10�3 0.01319 3.9 0.06069 18.1HCys HCys 5 6.6 2.7 (5.9) 4.16 � 10�5 5.24 � 10�4 12.6 9.74 � 10�3 234Cys HCys 6 0.20 3.7 (12.0) 1.75 � 10�6 1.60 � 10�5 9.1 2.0 � 10�4 114

a The reaction numbers correspond to those shown in Fig. 1.b One unit corresponds to 1 �mol of product formed min�1. The Km and Vmax values were determined as described under “Experimental Procedures” and reported in Table 1.c In reactions involving two substrates, the order of the Km values reflects the substrate order in the first column.d The values for the turnover numbers at varying concentrations of homocysteine and physiological concentrations of cystathionine (5 �M) and cysteine (100 �M) were obtainedas described under �Experimental Procedures� considering a ping-pong mechanism for the bimolecular reaction and the Hill coefficients (n) reported in Table 1.

e -Fold change refers to the change in v/�E� with respect to normal conditions (i.e. 10 �M homocysteine, which is assigned a value of 1 for each reaction).

TABLE 3The relative contributions of H2S-generating reactions at varyingconcentrations of homocysteine as predicted from kinetic dataanalysesThe reaction numbers correspond to those shown in Fig. 1. Values shown are thepercentage contribution of each reaction to net H2S production at each concentra-tion of homocysteine in the presence of 5 �M cystathionine and 100 �M cysteine.

Substrate Reactionnumber

10 �MHCys

40 �MHCys

200 �MHCys

% % %Cys 2 70.5 36.9 9.6Cys Cys 3 2.5 � 10�4 1.3 � 10�4 3.5 � 10�5

HCys 4 29.1 60.6 77.7HCys HCys 5 0.36 2.4 12.5HCys Cys 6 0.015 0.07 0.26

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SCHEME 1. Postulated reaction mechanisms for CSE-catalyzed reactions with cysteine and homocysteine.

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cleavage (0.008 s�1) and �12-fold higher than for homocys-teine cleavage (0.0034 s�1) (Table 2). Under these conditions,the �,�-elimination of cysteine catalyzed by CSE (Fig. 1, reac-tion 2) is the major source of H2S, accounting for �70% of itsproduction, whereas the �,�-elimination of homocysteine(reaction 4) accounts for �29% (Table 3 and Fig. 6).The normal range for plasma total homocysteine concentra-

tion in humans is �6–15 �M and is �25-fold lower than theconcentration of total cysteine (�250�M) (34). In patients withhyperhomocysteinemia, plasma homocysteine levels can rangefrom 20–50 �M (inmoderate hyperhomocysteinemia) to 100�M (severe hyperhomocysteinemia) (35). Since CSE catalyzeshomocysteine-dependent production of H2S, we have simu-lated the effect ofmoderate (40�M) and severe (200�M) hyper-homocysteinemic conditions, which boost the ratio of homo-cysteine/cysteine, on H2S biogenesis. As expected, H2S derivedfrom homocysteine-dependent reactions increased propor-tionately with the grade of hyperhomocysteinemia (Table 3 andFig. 6). The reaction displaying the greatest sensitivity to hom-ocysteine concentrations was the condensation of 2 mol ofhomocysteine to give homolanthionine catalyzed by CSE (Fig.1, reaction 5), which increased �230-fold at severely elevatedhomocysteine concentrations (Table 2). Under these condi-tions, homocysteine rather than cysteine becomes the pre-ferred source for CSE-derived H2S.The sensitivity of the CSE-catalyzed �-replacement reaction

to homocysteine suggests that homolanthionine, which isexpected to be more stable than H2S, could be a suitablebiomarker for this reaction. Indeed, homolanthionine wasreported in urine samples from homocystinuric patients nearly4 decades ago, when it was proposed to be derived from hom-ocysteine or homoserine metabolism (36). The first report ofbiologically derived homolanthionine dates back to 1963 in amutant strain of E. coli (37), and the accumulation of this com-pound was later reported in other organisms (38, 39). InCorynebacterium glutamicum, homolanthionine is an interme-diate in a novel pathway for isoleucine synthesis (40). Homol-anthionine formation by both rat and human liver CSE by thecondensation of homocysteine and homoserine has beenreported (41). Our study reveals that homolanthionine is gen-erated by the condensation of two homocysteinemolecules in areaction catalyzed by CSE, thus linking the origin of homolan-thionine to homocysteine metabolism and CSE. Homolanthi-onine could potentially be catabolized by reversal of the CSEreaction, and the metabolic fate of this compound needs to beevaluated.Our results suggest that under hyperhomocysteinemic con-

ditions, H2S productionmay be enhanced and could contributeto the associated cardiovascular pathology. In an in vivomodelfor myocardial ischemia-reperfusion, a U-shaped H2S dosedependence curve was observed, with the cardioprotectiveeffect of H2S decreasing at higher concentrations (7). In a ratmodel of stroke, administration of high NaHS levels increasedinfarct volume (42), and H2S was found to be proinflammatoryin a mouse endotoxic shock model (8). Mutations in CBS arethe most common cause of severe hyperhomocysteinemia incomparison with defects elsewhere in the pathway (e.g.methi-onine synthase, methyltetrahydrofolate reductase, andmethio-

nine synthase reductase). In homocystinuric individuals withCBS deficiency, CSEmay be themajor source of H2S. Our stud-ies suggest that inhibition of CSE in hyperhomocysteinemicindividuals could be a useful strategy for attenuating the attend-ant cardiovascular pathology seen with this disease.Lanthionine is another novel sulfur-containing amino acid

that is a by-product of H2S production by CSE (Fig. 1, reaction3). Although this thioether is a component of the class of pep-tide-containing antibiotics known as lantibiotics (43), its role, ifany, in mammalian biology is not known. The presence of thecyclic lanthionine ketamine compound has been reported inbovine brain (44), where it has been shown to bind with highaffinity (58 nM) to membranes, suggesting a possible role forthis compound in the central nervous system (45). CBS cancatalyze the synthesis of lanthionine from either cysteine orcysteine and serine (46). Our results demonstrate that CSE canalso catalyze the synthesis of lanthionine from cysteine. Therelative importance of CBS versus CSE in lanthionine produc-tion awaits further elucidation.In conclusion, our study reveals the relative importance of

cysteine- versushomocysteine-dependent reactions toH2S bio-genesis catalyzed by CSE. Since these CSE-dependent routesforH2S generation represent side reactions relative to its role inthe transsulfuration pathway, it is critically important to under-stand how these enzymatic reactions are regulated so that thesame catalyst can serve dual roles. In some cell types, such asvascular endothelial cells, the transsulfuration pathway is notintact, since CBS is reported to be absent (47). Since H2S is asignaling molecule, it is to be expected that its generation isregulated, and Ca2-calmodulin has been reported to activateCSE (20). Under hyperhomocysteinemic conditions, H2Shomeostasis may be dysregulated and could contribute to thecardiovascular etiology associated with this disease.

Acknowledgments—We thank Dr. Ashraf Raza and Ron Cerny (Uni-versity of Nebraska, Lincoln) for help with the mass spectrometricdata andDr. Dave Ballou (University ofMichigan) for helpful discus-sions on the kinetic simulations.

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Biogenesis of H2S from Cysteine and Homocysteine

11612 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 17 • APRIL 24, 2009

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Ruma BanerjeeTaurai Chiku, Dominique Padovani, Weidong Zhu, Sangita Singh, Victor Vitvitsky and

HyperhomocysteinemiaMetabolites Lanthionine and Homolanthionine and Is Responsive to the Grade of

-Lyase Leads to the Novel SulfurγS Biogenesis by Human Cystathionine 2H

doi: 10.1074/jbc.M808026200 originally published online March 4, 20092009, 284:11601-11612.J. Biol. Chem. 

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