THE JOURNAL OF BIOLOGICAL CHEA~TRY Vol. 240, No. 6, June 1865

hinted in U.S.A.

Enzymatic Synthesis and Cleavage of Cystathionine

in Fungi and Bacteria

COLETTE DELAVIER-KLUTCHKO* AND MARTIN FLAVIN

From the Laboratory of Biochenaist,ay, National Heart Institute, National Institutes of Health, Bethesda, Maryland 20014

(Received for publication, February 8, 1965)

Transsulfuration, the transfer of sulfur between cysteine and homocysteine, has been most thoroughly studied in mammalian liver, where it has been found to occur only in the direction homocysteine + cysteine, through the mediation of two separable pyridoxal phosphate enzymes, catalyzing Reactions 1 and 2

(1, 2).

Serine + homocysteine + Hz0 + cystathionine

p Replacement

(1)

Cystathionine + Hz0 --j cysteine + a-ketobutyrate + XH, (2)

y Elimination

In this paper we report on the reactions and enzyme components of transsulfuration in Neurospora crassa, Saccharomyces cerevisiae, Escherichia co& and Salmonella typhimurium, and show that the process is reversible in the higher fungi, and irreversible, in the direction opposite to that of mammalian tissues, in the bacteria.

Recently, we have reported the isolation of two cystathionine cleavage enzymes from Neurospora (3), one catalyzing predomi- nantly Reaction 2, the second Reaction 3.

Cystathionine + Hz0 + homocysteine + pyruvate + NH3 (3)

p Elimination

Yeast is essentially similar, whereas with the aid of unequivocal assays the bacteria can be shown to contain only one enzyme, catalyzing Reaction 3. Similarly, Reaction 1 is present in extracts of both fungi, and absent from both bacteria. We shall also report here the details of a preliminary communication (4) showing the bacterial synthesis of cystathionine from cysteine and the succinic ester of homoserine (Reaction 4).

0-Succinylhomoserine + cysteine +

cystathionine + succinic acid (4)

y Replacement

This reaction can not be demonstrated in the fungi, although indirect evidence has long indicated that Neurospora can syn- thesize cystathionine from cysteine (3). The implication of three genetic loci in the latter process in Neurospora (5), as against two in Salmonella (6), is a further indication of another generalized difference between the bacteria and higher fungi.

* Visiting scientist at the National Institutes of Health. Pres- ent address, Institute de Biologie Physico-chimique, Paris, France.

EXPERIMENTa4L PROCEDURE

Microbial Strains and Culture &ledia-Neurospora strains me-2 (P162) and me-7 (K79) have been described (3) ; me-3

(FGSC 502) and me-5 (FGSC 140) were obtained from the Dartmouth Fungal Genetics Stock Center. The yeast was a fresh local bakers’ yeast. The mutants derived from S. typhi- murium Lt-2 wild-type were (6): me-E (47), responding only to methionine; me-C (30), responding also to homocysteine; and me-B (16) and me-8 (15), both responding also to cystathionine. E. coli 26/18 was derived from wild-type W, and its nutritional responses were the same as those of Salmonella me-C. E. coli 2105 was from wild-type K12 (AB 1172), and its nutritional responses were the same as those of Salmonella me-E. P76/2 was also from type K12; it was not a methionine auxotroph, but was norleucine resistant and genetically derepressed in the methionine pathway.1

Culture media for Neurospora were as previously described (3). With the exception of P76/2, which was grown on nutrient broth, bacterial stocks were maintained on agar slants of Cold Spring Harbor medium A, supplemented, for the mutant strains, with 0.13 pmole of nL-methionine per ml. The medium A contains (in grams per liter): K2HPOI, 10.5; KH2P04, 4.5; sodium citrate.5Hz0, 0.5; (NH&Sod, 1.0; MgSO+ 0.05; and glucose, 2. For the preparation of bacterial extracts, 500 ml of the same liquid medium, in 2-liter Erlenmeyer flasks, was inocu- lated with 10 ml of an overnight liquid culture, and the flasks were incubated at 37” on a rotary shaker until reaching late exponential growth.

Many methionine derivatives were tested with the bacterial mutants, in attempts to derepress enzyme formation by providing nutrients which would only slowly yield L-methionine during growth. These included nL-methionine methyl ester, DL-

methionine methyl sulfonium chloride, n-methionine, N-acetyl- nL-methionine, L-methionine amide, glycyl-nL-methionine, DL-

methionine dl-sulfoxide, and DL-alanyl-DL-methionine. The last named compound gave the only favorable result (see Table III), a 2-fold increase in an enzyme level, associated with an increase in doubling time from 40 to 100 min. S-Methyl-L- cysteine did not support the growth of Salmonella me-E (47).

To determine the nutritional responses of Salmonella mutants to succinyl derivatives of homoserine (see Table VI), 30 ml of minimal medium with the appropriate supplement, was inocu- lated with 1 ml of an overnight culture grown with 0.03 pmole of nL-methionine per ml, a limiting amount. Growth responses

1 G. N. Cohen, personal communication.

2537

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2538 Synthesis and Cleavage of Cystathionine Vol. 240, No. 6

were measured turbidimetrically in 125-ml Bellco Nephalo flasks with a Klett-Summerson calorimeter and No. 66 filter. Nutritional responses similar to those shown in Table VI were also obtained on solid medium, and in liquid medium acidified to pH 5. Growth responses of Neurospora mutants were deter- mined by inoculating 25 ml of minimal medium (3), appropriately supplemented, with a small amount of conidial growth from a slant. dfter 3 days on a rotary shaker at 30”, the mycelium was filtered out, dried in a vacuum desiccator, and weighed. In all casts, the 0-succinyl-nn-homoserine and N-succinyl-nn-homo- serine were sterilized by Millipore filtration and added after the growth flasks had been autoclaved.

Enzyme Preparations-A lyophilized sample of crystalline rat liver cystathionine y-cleavage enzyme was obtained from Dr. D. M. Greenberg (7). Neurospora cystathionine y-cleavage enzyme was purified through Steps 3 or 4b as previously described (8). The remaining enzyme preparations used in this work were dialyzed crude extracts, prepared in various ways.

Yeast cells were disrupted with liquid nitrogen (9) ; after evaporation of nitrogen the homogenate was diluted with an equal volume of cold water and stirred for 1 hour at O”, the pH being maintained at 7.5 by addition of KHCOS. Dialysis in this case, and unless otherwise specified in all cases, was overnight at 2” against 0.01 i\f potassium phosphate, pH 7.5, containing low4 M

P-mcrcapt.opropionate and 2 X 1O-5 M pyridoxal-P. More protein could be extracted from yeast with a Branson model LST5 sonifier. Cells were suspended in a stainless steel con- tainer, in a large, stirred ice-water bath, in an equal part of 0.05 M potassium phosphate, pH 7.5, containing 10e3 M P-mercapto- prol)ionate and 2 X lop5 M pyridoxal-P. The suspension was sonified for a short period (3 to 5 min for 40 ml) at a setting of 7, and then centrifuged for 30 min at 14,000 rpm in an International HRl rentrifuge at 0”. In attempts to resolve cystathionine cleavage enzymes of yeast, dialyzed ultrasonic extract was diluted to a llrotein concentration of 10 mg per ml, and the protein was precipitated in six successive fractions between 25 and 75% saturation with ammonium sulfate (3). After freezing and thawing the original dialyzed extract a large portion of inactive protein precipitated, with an increase of specific activity for cystathionine cleavage (see below) from 0.0015 to 0.0075.

Neurospora was extracted with alumina or glass beads as previously described (3) ; good extraction could also be obtained with the Branson sonifier, after dispersing the mycelium in the buffer in a Waring Blendor.

Disruption of bacteria either with the sonifier, or by passage through a French pressure cell, gave similar results. A dialyzed extract of Salmonella me-A (15) was resolved for pyridoxal-P in several ways. Apoenzyme 1 was prepared by incubating extract with 0.1 M hydroxylamine at pH 7 for 30 min at 25”. Protein was precipitated with neutral saturated ammonium sulfate, washed three times with cold 0.9 saturated ammonium sulfate, dissolved in 0.1 M potassium phosphate, pH 7.3, con- taining 10e3 M P-mercaptopropionate and 10-4 M EDTA, and dialyzed against a solution containing 10e4 M of each of the latter in 0.05 M potassium phosphate, pH 7.3. Apoenzyme 2 was prepared by making an extract 0.8 M in potassium phosphate, pH 5.4, and incubating for 100 min at 37”. Protein was pre- cipitated with solid ammonium sulfate, and the precipitate was washed, dissolved, and dialyzed in the same way as Apoenzyme 1.

Assays of Elimination Reactions -Assay procedures for cysta- thionine cleavage enzymes (Reactions 2 and 3) have been de-

scribed (3, 7), which involve measurements of the rate of libera- tion from cystathionine of mercaptoamino acids or of ac-ketoacids. Total mercaptoamino acid liberation is measured by adding an aromatic disulfide, ArSSAr,2 to the enzyme reaction mixture, and following the formation of colored aryl mercaptan; cysteine and homocysteine are measured individually by a more laborious procedure utilizing radioactive EM. Neither procedure could be used for Neurospora P-cleavage enzyme, which was inhibited by both reagents (3). However, the bacterial /3 enzyme was found not to be inhibited, and was assayed in both ways. It is not clear from the results below whether or not the /3 enzyme from yeast can be assayed by these procedures.

The alternative assay for cleavage enzymes utilizes lactic dehydrogenase to measure both the rate of a-ketoacid liberation and the proportions of a-ketobutyrate and pyruvate (3). It can be used for all cleavage enzymes, but has several limitations. Accurate rate measurements are difficult to obtain with crude extracts because the a-ketoacids undergo further decomposition. Also, the mercaptoamino acids liberated, after air oxidation to the disulfide forms, become substrates for cystathionine cleavage enzymes and in turn liberate a-ketoacids (3, lO).3 In order to block disulfide formation, we either added iodoacetate or EM to the reaction mixture, or used a helium gas phase, although it is not feasible to exclude all oxygen with the latter (3). A unit of enzyme activity is defined in all cases as the amount decomposing 1 pmole of cystathionine in 1 min under standard conditions.

Assays of Replacement Reactions-Cystathionine formation from homoserine + cysteine (y synthetase), or from serine + homocysteine (p synthetase), was measured by determining the incorporation into it of the appropriate 14C-hydroxyamino acid or a%-mercaptoamino acid. These procedures were used mainly to demonstrate the presence or absence of the enzyme activity in extracts, although proportionality to extract protein was shown with the Neurospora /3 synthetase. Since extracts contain cysta- thionine cleavage enzymes, a conclusive negative result could only be obtained by adding carrier cystathionine to the reaction mixture and showing that it had not all been consumed at the end by ninhydrin reaction on chromatograms.

These assays and those below required an anaerobic gas phase, which was obtained by slowly bubbling helium through the reaction mixtures, in l-ml centrifuge tubes, for 15 min before making the final additions of enzyme and mercaptoamino acid. The latter were treated separately in the same way, the gas stream passing over the surface of the enzyme solution. For longer anaerobic storage, tubes were flushed with helium by inserting two small guage syringe needles through a rubber serum vial cap.

Reactions were terminated by precipitating protein with 0.1 volume of 1.5 M perchloric acid and, depending on the components of the mixture, some of the supernatant solutions were then desalted with 0.5- x 2-cm columns of Dowex 50 (H+). After applying the sample, the columns were eluted with water until the pH became neutral, and then amino acids were eluted with N NH40H. When the eluate became alkaline, two or three more column volumes were collected. The combined ammonia elu- ates were evaporated in a vacuum over H2SG4. Residual am-

2 The abbreviations used are: ArSSAr, 5,5’-dithiobis-(2-nitro- benzoic acid) ; EM, N-ethylmaleimide.

3 Unpublished results. The E. coli cystathionine cleavage enzyme has been purified 500.fold over the wild-type W level. Studies of its properties and substrate range (8) will be reported elsewhere.

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June 1965 C. Delavier-Klutchko and M. Flavin 2539

monia was removed by adding 1 pmole of NaOH and repeated evaporation. If the mixture contained 35S, the residue was now treated for 30 min at 25” with performic acid (30% H202-90% formic acid; 1: 9, to allow chromatographic separation of cysteic and homocysteic acids from “oxidized cystathionine” (a single spot was always observed, but it is not known to what extent the product was the sulfoxide or sulfone).

Cystathionine was now isolated by descending chromatography on Whatman No. 1 filter paper in Solvent 1, tert-butyl alcohol- formic acid-water (70 : 15 : 15), or by ascending chromatography in Solvent 2, propyl alcohol-l 1 N HCl-water (60 : 20 : 10). Radio- active components were located by exposing the chromatograms to Eastman single-coated blue x-ray film. The RF values are indicated in the figures under “Results.” Like cystine, cysta- thionine chromatographs poorly in most solvent systems be- cause of its insolubility, tending to streak and migrate to variable positions depending on the amount present. When present in large amounts oxidized cystathionine sometimes remained at the origin in Solvent 1. Elution was usually possible with dilute acetic acid, but sometimes required 2 N HCl, which caused some destruction of oxidized cyst’athionine.

Assays of Reactions Involving 0-Succinylhomoserine-The discovery that the same enzyme catalyzing Reaction 4 also

0-Succinylhomoserine + Hz0 + succinic

acid + a-ketobutyrate + NH3 (5)

catalyzed Reaction 5 in the absence of cysteine (4) provided a convenient means for assaying the amount of enzyme through the rate of a-ketobutyrate formation. Because of the high level of this enzyme in bacteria, and because mercaptoamino acids are not products, the assay for a-ketobutyrate gave an acrurate measure of the amount of enzyme in crude extracts, and was not subject to the limitations mentioned above for the elimination reactions from cystathionine. Formation of cystathionine in Reaction 4 was studied by the chromatographic method described above, and also by following the disappearance of O-succinyl- homoserine and of cysteine.

0-Succinylhomoserine was determined by alkaline hydroxyla- mine assay after oxidative deamination with bromine. If the amino group is not first removed this ester gives a negative hy- droxylamine test (4). An aliquot of a deproteinized reaction mixture, containing 0.5 to 1 pmole of 0-succinylhomoserine, was added to a glass-stoppered centrifuge tube with enough water to make the final volume 1 ml. To this 0.1 ml of M potassium ace- tate, pH 5.0, was added, followed by 0.05 ml of a solution of 4 M

bromine in 4 M sodium bromide. The stoppered tubes were shaken for 30 min at 30”. Helium was then bubbled through t,he solution for 3 to 4 min in a hood, to remove most of the bromine, and the remainder was reduced with M sodium sulfite, 0.03 ml or enough to decolorize. After adding 0.2 ml of freshly prepared 1: 1 mixture of 4 M hydroxylamine hydrochloride and 7 N NaOH, the solutions were incubated at 25” for 3 min. Then 0.1 ml of 8 N HCI was added, followed by 0.1 ml of 20% FeC13 .6H,O in 0.2 N HCl. The molar absorbance of 0-succinylhomoserine at X640 was 700. Components of the original reaction mixture may yield elevated blanks or interfere with the assay (11). It was desirable to run two controls for every determination. A mock original reaction mixture to which no 0-succinylhomoserine was initially added, and an aliquot of the same to which 1 pmole of O-succinyl- homoserine was added after deproteinization.

This disappearance of cysteine from reaction mixtures was determined by nitroprusside assay with and without cyanolysis (lo), since, despite all efforts to make the reaction vessels anaero- bic, some oxidation to the disulfide always occurred. Reliable results were obtained only when the latter occurred to a small extent.

i?fateriaZs-The preparation has been described of O-succinyl- nL-homoserine (4), N-succinyl-nL-homoserine (4), and DL-

homoserine-2-‘4C (7), as has the source of cystathionine and other amino acids (3). L-Serine-3-14C was obtained from Nuclear- Chicago Corporation; nL-homocysteine-35S-thiolactone hydro- chloride was from Volk Radiochemical Company, and was delactonized for use by heating a solution for 3 min at 100” with 2 eq of NaOH. DL-Cystine-35S (20 to 40 MC per pmole) was from Nuclear-Chicago Corporation. For use, 4 pmoles were dissolved in 0.2 ml with the aid of a minimal amount of NaOH. Sodium hydrosulfite, 0.02 ml of 0.4 M adjusted to pH 7, was added, and the solution left at 25” for 30 min. Reduction was incomplete. However, an indeterminate further reduction took place during the enzyme incubation, by disulfide interchange with carrier L-cysteine (Merck) which was added separately.

RESULTS

Cystathionine Cleavage Enzymes of Yeast and Bacteria-The ability of yeast to decompose cystat’hionine by y elimination (Reaction 2) and fi elimination (Reaction 3) was investigated by measuring the proportions of Lu-ketobutyrate and pyruvate liberated (Table I). Cystathionine was decomposed relatively slowly by yeast extracts, as compared to Neurospora extracts (3), and the u-ketoacids formed usually underwent rapid decomposi- tion. A number of enzyme preparations were examined until by chance some were found in which oc-ketoacids were sufficiently stable to give significant results. Bot’h pyruvate and a-keto- butyrate were liberated (Table I); it was not possible to separate the activities by ammonium sulfate fractionation. In Xeuro- spora, two cystathionine cleavage enzymes can be partially separated in this way; the P-cleavage enzyme is unstable and is inhibited by EM and ArSSAr; the y-cleavage enzyme catalyzes a small amount of Reaction 3, while liberating preponderantly a-ketobutyrate (3). Further work is required to determine whether the same situation exists in yeast or not. The results of Table I indicate that a preponderance of pyruvate was formed only by a fresh extract obtained by a mild procedure (Experi- ment 2), and in the absence of EM or iodoacetate. Rates sug- gest the latter may have been due not only to inhibition of a P-enzyme by EM (and, unlike Neurospora, iodoacetate), but also to inhibition of a y-enzyme by the product, cysteine, ac- cumulating in the anaerobic incubation (8). As with Neurospora, significant small amounts of pyruvate were formed by all enzyme fractions.

Extracts of wild-type Salmonella and E. coli W were found to decompose cystathionine exclusively by Reaction 3 (12). These results confirm other reports of E. coli (13. 14) which had reached the same conclusion, although with methods less suited to detect the formation of small amounts of a-ketobutyrate and cysteine. Unlike the cystathionine P-cleavage enzyme of Neurospora, the bacterial enzymes were not inhibited by EM or aromatic disulfides (Table II). It was therefore possible to incorporate radioactive EM into the reaction mixture, and by chromato- graphing the labeled homocysteine and cysteine adducts formed (7), to detect the liberation of very small amounts of cysteine.

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2540 Xynthesis and Cleavage of Cystathionine Vol. 240, No. 6

TABLE I Cystathionine cleavage by extracts of yeast

The incubation systems were either with a gas phase of helium, without added sulfhydryl traps, as described in the text, or aerobic

in the presence of 0.01 M EM or iodoacetate. Either a 0.03 M potassium phosphate, pH 7.5, or a 0.03 M potassium pyrophosphate, pH

8.0, buffer was used, as indicated. All reaction mixtures contained n-cystathionine, or pyruvate, or a-ketobutyrate, as indicated, and 0.2 pmole of pyridoxal-P, in a final volume of 1 ml, and were incubated at 30”. In Experiment 1 the reaction mixtures contained 4.6

mg of protein from a liquid nitrogen extract of yeast, specific activity by ArSSAr assay was 0.0016, and the incubation was for 60 min. In Experiment 2 the mixtures contained 8 mg of protein from a fresh sonic extract, ArSSAr specific activity was 0.0015; incubated 135 min. In Experiment 3 the mixOures contained 8 mg of protein from a sonic extract stored frozen for 1 month, ArSSAr specific activity

was 0.0076; incubated 120 min.

system PH

Experiment 1 Helium

EM Experiment 2

Helium

Helium EM EM

EM Iodoacetate Iodoacetate

Iodoacetate Experiment 3

EM EM EM

Iodoacetate Iodoacetate Iodoacetate Iodoacetate Iodoacetate Iodoacetate

7.5 Cystathionine 2 0.039 0.090

7.5 Cystathionine 2 0.040 0.110

7.5 Cystathionine 2

7.5 Pyruvate 0.86 7.5 Cystathionine 2 7.5 Pyruvate 1.03

7.5 a-Ketobutyrate 0.86 8.0 Cystathionine 2 8.0 Pyruvate 0.90 8.0 c+Ketobutyrate 0.81

7.5 Cystathionine 2 0.057 0.57 7.5 Pyruvate 2.1 1.8 0

7.5 a-Ketobutyrate 2.3 0.2 1.8

7.3 Cystathionine 2 0.11 0.80 7.3 Pyruvate 2.2 2.3 0 7.3 a-Ketobutyrate 2.5 0.15 2.2 8.0 Cystathionine 2 0.08 0.28

8.0 Pyruvate 1.8 2.0 0 8.0 a-Ketobutyrate 2.3 0.15 2.3

Incubation

* Approximate rate of formation of total a-keto acid.

Xone was found.3 That the apparent formation of 7% of (Y- ketobutyrate by E. coli extract (Table II, Experiment 2) was within the analytical error, is shown by the apparent formation of the same per cent of pyruvate by liver enzyme in a control experiment under the same conditions (Table II, Experiment 3). Results with labeled EM have shown that liver enzyme forms only cysteine (7).

Because it is not inhibited by aromatic disulfides, the bacterial cystathionine P-cleavage enzyme can be accurately measured in crude extracts by the ArSSAr assay. Table III shows the results

of a survey of several bacterial strains. Some of the mutant strains blocked in steps of methionine biosynthesis had enzyme levels higher than those of the parental wild-types, even though they had been grown in the presence of repressor end product (15); the maximum further increase obtained by attempts to derepress was 2-fold.

Bs previously reported, the enzyme was absent from Xalmonella me-C(30) (12) and E. coli 26/18 (15). Both these mutants were relatively unstable. When low enzyme levels were observed (Table III) the population could be shown to contain a proportion of revertants. A different characteristic of enzyme production by Salmonella me-C(30) is shown in Table IV. At 25” this mutant grew on minimal medium and formed small amounts of

Amount

Products

Pyruvate a-K&o- butyrate

pmoles

0.22

0.75 0.081 1.2

0.021 0.90

0.11

0.26

0.93 0.062

0.87

Pyruvate

%

30

27

67

24 87

31 37

21

24 117 108

26 5 100 107

10

12

22

18 85 77

26 107

89

10

-

110 100

Rate (as per cent of that observed y ArSSAr assay)*

active enzyme in the presence or absence of exogenous methio- nine. This was not due to reversion. These cells again required

methionine for growth, after the first transfer, when subcultured at 37”.

Cystathionine Xynthesis by ,8 Replacement-Evidence that Neurospora extracts contain an enzyme (cystathionine 0 syn- thetase) catalyzing the synthesis of cystathionine from serine and homocysteine (Reaction 1) is shown in Fig. 1. Strips 1 to

3 show the effect of increasing protein concentration on the amount of serine-3-1”C incorporated into cystathionine. In- corporation was proportional at lower enzyme levels. The smaller accumulation of cystathionine at the highest enzyme level (Fig. 1, Strip 3) might have been due to the presence of cleavage enzymes, since the presence of a pool of unlabeled cysta- thionine (Strip 6) again increased the labeling of cystathionine. An apparent stimulation by ATP (Strip 7), at the high enzyme level, was presumably an indirect effect, since the @ synthetase reaction was catalyzed by purified enzyme fractions (see below)

that could not have contained ATP. A requirement for pyri- doxal-P is suggested by the inhibition by hydroxylamine (Strip 5). Finally, no cystathionine was formed if homocysteine was omitted (Strip 4). Substitution of cysteine led to incorporation of a very small amount of label int’o a compound, presumably

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-June 1965 C. Delavier-Klutchko and M. Flavin 2541

TABLE II

Cystathionine cleavage by extracts of bacteria

Incubation systems and conditions as in Table I. In Experiment 1 the mixtures contained 3.7 mg of protein from a sonic extract of Salmonella, ArSSAr specific activity, 0.0044, and the incubation was for 100 min. Experiment 2, 2.6 mg of protein from French press extract of E. coli, specific activity, 0.011; rates determined in 30.min incubation; product proportions in 130 min. Experiment 3,

control incubations with crystalline liver cystathionine cleavage enzyme; 0.3 mg of protein, specific activity, 0.094; incubation time, 130 min.

system

Experiment 1. Salmonella Lt.2 wild-type

Helium EM Iodoacetate

Experiment 2. E. coli W wild-

type EM

EM EM Iodoacetate

Experiment 3. Rat liver cysta- thionine cleavage enzyme

EM Iodoacetate

Incubation Products

PH Addition Amount Pyruvate

7.3 Cystathionine 2 0.63 7.3 Cystathionine 2 0.70 7.3 Cystathionine 2 0.74

7.3 Pyruvate 0.416 0.402

7.3 Cystathionine

7.3 Pyruvate 7.3 a-Ketobutyrate 8.0 Cystathionine

0.73

1.09

1.56

7.3 Cystathionine 8.0 Cystathionine 8.0 a-Ketobutyrate

2 1.09

0.87 2

2 2 0.47

0 0.08

* Approximate rate of formation of total a-keto acid.

L L

lanthionine, with the same RF as cystathionine. Incubation of

labeled serine or other amino acids (especially 3%mercapto-

amino acids) with crude extracts leads to the formation of traces

of many radioactive products. Reaction Mixture 4 (Fig. 1) contained a radioactive unknown which chromatographed like cystathionine in Solvent 1, but was resolved from the latter in Solvent 2.

could not decompose cystathionine; the extract did not catalyze serine-dependent disappearance of homocysteine, or incorpora- tion of serine-*4C into cystathionine.

The identity of the reaction product is supported by the fol- lowing results. The labeled cystathionine was eluted from the second (upper) chromatogram of Strips 1, 2, 3, and 7 of Fig. 1. After treatment of an aliquot with Raney nickel (16) the only radioactive product detectable in chromatograms corresponded to alanine. Treatment of a second aliquot with excess purified Neurospora cystathionine y-cleavage enzyme in the presence of unlabeled EM (7), yielded labeled cysteine-EM adduct. Ap- proximate quantitative measurements of the amounts of sub- strates consumed and product formed, in reaction Mixtures 1 to 3 of Fig. 1, are shown in Table V. In Mixture 3, the measure- ment of homocysteine remaining at the end of the reaction may include some cysteine formed by cystathionine cleavage.

Some early observations had suggested that a single enzyme might participate in more than one of the reactions mediating transulfuration in Neurospora (17); such a possibility is also plausible from the standpoint of pyridoxal-P catalysis (17). The enzyme catalyzing p replacement is not identical with the cysta- thionine P-cleavage enzyme since it was present in mutant me-d (P162), which lacks the latter (Fig. 2, Strip 4) (3). There are no Neurospora mutants lacking y-cleavage enzyme (3), but the results of Strips 1 to 4 in Fig. 3 indicate that it, also is not identi- cal with the enzyme catalyzing B replacement. Although the purified y-cleavage enzyme fraction still catalyzed some /3 replacement (Fig. 3, Strip 3), the ratio of cleavage to replace- ment was higher with this fract.ion than with crude extract. Crystalline liver y-cleavage enzyme also did not form cysta- thionine from serine and homocysteine (Fig. 2, Strip 6). Suffi- cient carrier cystathionine was present during these reactions t’o ensure that some remained at the end.

The same results obtained with Neurospora were also obtained Cystathionine Synthesis from 0-Succinylhomoserine and Cysteine with yeast extract. Formation of labeled cystathionine from (y Replacement) in Bacteria-From indirect evidence the syn- serine-1% was due to a net synthesis; it required addition of thesis of cystathionine from homoserine and cysteine (17) had homocysteine (Fig. 2, Strip 2), but not of a pool of cystathionine been postulated years ago by a number of investigators to occur (Strip 3). Strip 7 of Fig. 2 shows formation of labeled cysta- in E. coli (18) and Neurospora (19). However, the first direct thionine, by Neurospora extract, from homocysteine-“% and evidence for the existence of this reaction appeared in a series of unlabeled serine. Strip 5 of Fig. 2 illustrates the negative brief reports by Rowbury 3 years ago (15). The reaction was results obtained in many experiments with extracts of wild-type reported to be catalyzed by extracts of E. coli supplemented E. coli and Salmonella. Cystathionine B synthetase also could with succinate, ATP, coenzyme A, and glucose. Sequential not be detected in an extract of Salmonella me-C(30), which incubations with extracts of E. coli methionine auxotrophs in-

or-Keto- butyrate

0 0 0

0

0.87

0.12

0.14 1.07 0.43

Pyruvate

7%

100 100 100

100

93

0 7

Recovery

5%

97

100 100

92

Rate (as per cent of that observed

by ArSSAr assay*)

39 41 43

70

130

100

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2542 Synthesis and Cleavage o;T Cystathionine Vol. 240, No. 6

TABLE III

Enzymatic reaction rates in bacterial el tracts

cysteine and the second extract, the latter was converted to a product chromatographically similar to cystathionine.

We were not able to show the occurrence of the over-all reac- tion, under the conditions briefly described by Rowbury, with extracts of Neurospora or yeast (3), or wild-type E. coli. With extracts of E. coli 26/18 incorporation of labeled homoserine or cysteine into a product chromatographically similar to cysta- thionine could be shown, but the extent of the reaction was very small.

We then synthesized the two obvious candidates for the uni- dentified intermediate of Rowbury, the succinic ester and amide of homoserine (4). We shall now describe t,he details of some studies of the nutritional properties and enzymatic reactions of these two compounds (4). Recently, Rowbury and Woods have also reported considerable evidence indicating that the natural intermediate is 0-succinylhomoserine (20).

In Salmonella, two genetic loci have been implicated in the formation of cystathionine from cysteine (6), as indicated by the ability of mutants mapping within them to respond t,o methio- nine, homocysteine, or cystathionine, but not to cysteine (Table VI). Strain me-A (15) grew with a wild-type doubling time on the former two, while growth of me-B(16) was slower on all three. If 0-succinyl- or N-succinylhomoserine were an intermediate in methionine biosynt,hesis, it was expected that one of these strains would show an appropriate growth response, particularly since some evidence had been reported that me-B could feed me-A

mutants (6). However, this was found not t’o be the case. Growth which occurred after a very long lag time, in the presence of t’hese compounds, was found to be due to reversion, as shown in the last column of Table VI. On subculture, these cells grew equally well in minimal medium as with methionine, although more slowly than the parental mutant in t,he latter case. The same negative results were obtained in other experiments in which the filter-sterilized homoserine derivatives were added by serial addition of aliquots over a B-hour period, or were added in varying amounts together with cysteine; there was also no growth response to either compound on solid medium or liquid medium acidified to pH 5.

In Neurospora three genet’ic loci, rather than two, have been implicated in the formation of cystathionine from cysteine (5). Representative mutants of these loci likewise did not respond to the succinyl derivatives of homoserine (Table VII).

However, when dialyzed extracts of E. coli 26/18 were supple- mented with cysteine-35s and 0-succinylhomoserine, there was extensive incorporation of label into a product chromatographi- tally indistinguishable from cystathionine (4). No cystathionine was formed from N-succinylhomoserine (4). Fig. 4, lower, shows some results of experiments of this t,ype with extracts of Sal- monella mutants. The format’ion of cystathionine was much more rapid than its cleavage, since the same amount accumulated with me-A(15) extracts as with those of me-C(30), which lacks cleavage enzyme (Strip 8). Cystathionine formation was pro- portional to me-d(15) protein concentration (Strips 1, 6, 5, and 4), and was completely inhibited by hydroxylamine (Strip 3). Extracts of me-B(16) did not catalyze any reaction (Strip 7).

Cystathionine formation by me-il(15) apoenzyme preparations was stimulated by pyridoxal-I’, but not by pyridoxal (Fig. 4, upper, Strips 1 to 4). rlTP did not effect the holoenzyme reac- tion (Strips 5 and 6), and cystine could not replace cysteine (Strips 7 and 8).

- Specific activities of extracts*

O-Succinyl- homoserine

Zystathio- consumption by line cleav- tge (Reac-

tion 3)

Dere- pressiont Bacterial strain

pmole/mg x min

0.0061 0.046

0.078

02 0.006

0.042 0.055 0 0064

0.010

0.4

0.023 0.16

Salmonella LT.2 wild Salmonella me-A

Salmonella me-A. Salmonella me-B. Salmonella me-C.

Salmonella me-E..... Salmonella me-E. E. coli W wild.. E. coli 26/l%.

E. coli K12 wild, E. coli 2105.......

E. coli P76/2.

0.004 0.008

0.015 0.005 0

0.005 0.010

0.015

0.001

0.030 0.079

0.083 -

* Reaction 3 was measured by ArSSAr assay. The assay conditions for Reactions 4 and 5 are described in the legend to Table VIII.

t Salmonella me-d (15) grown on medium A + 0.1 pmole per ml of cystathionine; me-E (47) on medium A + 0.07 Mmole per ml of DI,-alanyl-DL-methionine (see “Experimental Procedure”) ; P76/2 is genetically derepressed.

$ Sensitivity, l/300 of Salmonella me-A (15).

TABLE IV

Effect of growth temperature on cystathionine cleavage

enzyme activity of Salmonella me-C(30)

Cystathionine cleavage activity’ Growth

(absorb- ance at 650 mp)

Growth conditions

Medium Temper- ature

Strain LT.2 Broth............. 37” A. 37 A + methionine 37 A. 25

Strain me-C(30) Broth.. 37 A. 37 A + methionine. 37 A................. 25 A + methionine 25

Time ArSSAr a-Ketoacid aSSay assay

hr

22 0.66 0.0014

46 0.8G 0.0045 22 0.82 0.0025

28 0.83 0.0052

22 0.77 16 0

22 0.58 16 0.66 16 0.80

0. ooozt

0.0001~

0.0009

0.0011

* Dialyzed sonic extracts were assayed for Reaction 3 by the standard ArSSAr assay, and by the rate of CY ketoacid liberation in the helium system described in the legend of Table J.

t Not reliably measurable.

dicated that at least two enzymes were required. The first extract, in the presence of homoserine, succinate, and the co- factors, catalyzed the formation of an intermediate containing the elements of succinate and homoserine; in the presence of

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June 1965 C. Delavier-Klutchko and M. Flavin 2543

FIG. 1 (left). Cystathionine synthesis from serine and homo- cysteine (p replacement) by extracts of Neurospora. Photograph of radiograms obtained after paper chromatography of various enzyme reaction mixtures applied at Strips 1 to 7 at the origin (horizontal line, Zmer half) and developed with Solvent 1 (de- scribed in the text). The RF symbols are: C, cystathionine; S, serine. The area corresponding to cystathionine, from each reac- tion mixture, was elut,ed with 0.2 N acetic acid, and rechromato- graphed in Solvent 2 (upper haZf). Exposure times of the two chromatograms to the x-ray film were not the same. The eluate from reaction Mixture 6 was accidentally lost. The reaction mix- tures were incubated for 4 hours at 30” under helium, and con- tained, in a volume of 0.5 ml: potassium phosphate, pH 7.5, 20 hmoles; pyridoxal-P, 0.2 pmole; L-serine-3-14C, 2 Hmoles (0.2 pC per @mole); dialyzed sonic extract of wild-type Neurospora, ArSSAr cystathionine cleavage specific activity, 0.0007 (amount indicated below) ; and, except where indicated, 2 pmoles of n-homocysteine. Reaction Mixture 1 contained 1.4 mg of enzyme; 8, 2.8 mg of enzyme; S, 4.2 mg of enzyme; 4, 4.2 mg of enzyme without homo- cysteine; 5, 4.2 mg of enzyme + 1.5 pmoles of hydroxylamine; 6, 4.2 mg of enzyme + 2 Fmoles of carrier L-cystathionine; 7, 4.2 mg of enzyme + 1.5 pmoles each of ATP and MgC12. After de- proteinization with &volume of 1.5 M HC104, O.l-ml aliquots were applied directly to the chromatogram.

Since there is no convenient assay for cystathionine, quantita- tive studies of Reaction 4 (cystathionine y synthetase) were carried out by measuring the disappearance of cysteine and 0-succinylhomoserine from reaction mixtures. As shown in Experiment 1 of Table VIII, t’he two substrates disappeared from enzyme reaction mixtures equally until half the racemic ester had been consumed; neither substrate was consumed by extracts of Xalmonella me-B(16). The small consumption of substrates in the absence of enzyme, although within the analyti- cal error in this experiment, was real, since the ester underwent slow spontaneous rearrangement to N-succinylhomoserine under these conditions (4), and air could not be totally removed, to block oxidation of cysteine lo the disulfide. Pyridoxal-P was

FIG. 2 (right). Cystathionine synthesis by p replacement cata- lyzed by extracts of yeast, E. coli, Neurosporu me-d mutant, and liver cystathionine cleavage enzyme. General conditions as in Fig. 1; additional RF symbol: CO, oxidized cystathionine. Strip 7 shows the formation of radioactive cystathionine by a Neuro- sporu wild-type extract under conditions similar to those of Mix- tures 1 to 3 (Fig. l), except that the mixture contained nn-homo- cysteine-3% and unlabeled serine. After performic oxidation, the mixture was chromatographed in Solvent 1, oxidized cysta- thionine was eluted with 0.2 N acetic acid and was rechromato- graphed in Solvent 2 (Strip 7). The other strips are all from reaction mixtures chromatographed in Solvent 1 only, and not oxidized. Strips 4 to 6 are from reaction mixtures containing serine-3-‘4C and homocysteine, under conditions similar to Strips 1 to 3 of Fig. 1, with the following exceptions. The reaction mixtures contained 2.5 pmoles of carrier n-cystathionine, the incubation time was 60 min, and the enzyme preparations were: Strip 4, alumina (3) extract of A’eurospora me-2(P162), 5 mg; Strip 5, sonic extract of E. coli W wild-type, 2 mg; Strip 6, crystal- line liver cystathionine cleavage enzyme, 0.3 mg, ArSSAr specific activity, 0.04. Strips 1 to 3 are from reaction mixtures similar to Strips 4 to 6 except that the enzyme was 5 mg of a sonic extract of yeast; homocysteine was omitted from the reaction mixture of Strip 2, and carrier cystathionine from Strip 3; incubation time, 30 min.

required (Experiment 3), and ATP had no effect (Experiment 2). I f cysteine was omitted, half the added ester was still decomposed, but by a different reaction (y elimination, Reaction 5) yielding Lu-ketobutyrate (Experiment 4). Addition of cysteine totally inhibited cr-ketobutyrate formation from the ester. A small amount of pyruvate was formed from cysteine (probably after air oxidation, see below) ; this was part]ially inhibited by the ester. Homoserine itself yielded oc-ketobutyrate much more slowly (Table VIII) ; this reaction was only partially inhibited by cysteine. and homoserine did not inhibit pyruvate formation from cysteine. The extract used in these experiments did not contain cystathionine cleavage enzyme.

Experiment 5 of Table VIII shows that cystine inhibited LY-

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2544 Xynthesis and Cleavage of Cystathionine Vol. 240, n-o. 6

TABLE V

Stoichiometry of Neurospora cystathionine p synthetase reaction The amount of serine-3-14C incorporated was determined by

counting aliquots of eluates of the radioactive areas corresponding to cystathionine, after chromatography in Solvent 1 (Fig. 1). The amount of homocysteine consumed was determined by ti- tration with ArSSAr (10) of aliquots of the original reaction

mixtures of Fig. 1, at zero and final times. The amount of cysta- thionine formed was estimated in deproteinixed reaction mixtures. ArSSAr was added (reacting instantaneously with the homo-

cysteine present), followed by purified E. coli cystathionine &cleavage enzyme,3 and the amount of additional mercapto- amino acid liberated by the enzyme was followed at X412 (10).

For the last two assays, blanks were subtracted which were obtained with reaction mixtures similar to No. 3 of Fig. 1, but without serine.

w pmoles /moles pmoles

1 1.4 0.8 1.0 0.9 2 2.8 1.7 1.9 1.7 3 4.2 0.6 0.7 0.6

ketobutyrate formation from the ester very little. More pyru- vate was formed than from cysteine, and-the amount equaled the cystine consumed (less a small amount of pyruvate evidently formed from endogenous material in this experiment). These results are consistent with direct decomposition of cystine, by the bacterial extract, by P-disulfide elimination (10). The results of Table IX show that Reaction 5 also required pyridoxal-P, but not ATP, and that N-succinylhomoserine did not undergo this reaction.

Reaction 5, as well as 4, was not catalyzed by Salmonella me-B(16) extracts, as shown in Table III, which summarizes the relative rates of these reactions in various other bacterial extracts. With the same extract, cystathionine formation from O-succinyl- homoserine + cysteine was about 10 times faster than cY-keto- butyrate formation from 0-succinylhomoserine in the absence of cysteine. The rates of Reactions 5 and 3 were comparable, but the former was relatively higher in Salmonella than E. coli. In Salmonella me-A(15) and me-E(47) extracts, the rate increase over wild-type was also greater for Reaction 5 than 3. The rate of Reaction 5 in Salmonella me-A (15) grown on cystathionine (doubling time 80 min, Table VI) probably represents maximum derepression. When the doubling time of this strain was in- creased to 6 hours with an exponential gradient generator (21), the cell extract had the same specific activity.4

Cystathionine Synthesis by y Replacement in Yeast and Neuro- spora-In contrast to the bacteria, all attempts to show labeled cystathionine formation from 0-succinylhomoserine and cysteine- 3% by extracts of Neurospora or yeast have so far been unsuccess- ful. These experiments were carried out with a variety of ex- traction procedures, of supplements to the reaction mixtures, and with extracts of me-W, me-S, me-5, and me-7 mutants of Neurospora. An extract of a young (6 hour) conidial culture of Neurospora also gave negative results; the cystathionine y- cleavage enzyme activity of this extract was similar to that of the usual mycelial extracts. This cleavage enzyme, which as shown

4 M. M. Kaplan and M. Flavin, unpublished results.

above is also present in yeast, but absent from the bacteria, rapidly decomposes 0-succinylhomoserine by Reaction 5 (8). The bacterial cystathionine P-cleavage enzyme does not decom- pose 0-succinylhomoserine, since the latter is not decomposed by extracts of Salmonella me-B(16) which contain this enzyme (Table III), or by purified fractions of the cleavage enzyme.3 However, sufficient amounts of 0-succinylhomoserine were added to reaction mixtures to ensure that failure to observe cystathionine formation from it was not due to its decomposition by the fungal cystathionine y-cleavage enzyme.

Liver cystathionine cleavage enzyme has been reported to slowly incorporate labeled homoserine into cystathionine in the presence of cysteine (22). This result is confirmed in the experiment of Strip 5 of Fig. 3; Strip 8 shows that liver enzyme also catalyzed incorporation of cysteine-3% into cystathionine, in the presence of unlabeled homoserine. The reaction is thus

FIG. 3. Cystathionine synthesis from homoserine and cysteine by cystathionine r-cleavage enzymes purified from liver and Neurospora, and from se&e and homocysteine by the Neurospora y-cleavage enzyme. General conditions as in Figs. 1 and 2. The RF symbols are: HS, homoserine; CA, cysteic acid; C, cysta- thionine; S, serine; CO, oxidized cystathionine. Strips 1 to 4 are from reaction mixtures incubated 60 min at 30” and containing, in a final volume of 0.5 ml: potassium phosphate, pH 7.5,20pmoles; pyridoxal-P, 0.2 pmole; n-cystathionine, 2.5 hmoles; and n-serine- 3-W, 2 pmoles. Mixtures 1 and 3 also contained 2 pmoles of n-homocysteine. The enzyme preparations used were crude Step 1C (Strips 1 and 2) and purified (8) Step 3 (Strips 3 and 4) cystathionine r-cleavage enzyme from Neurospora me-d(P162); 2.3 mg of protein was added in each case, the ArSSAr specific activity of Step 3 being 10 times greater than that of Step 1C. The reaction mixtures of Strips 5 to 7 contained 6 pmoles of L- cysteine, 0.7 pmole of nn-homoserine-2J4C, and 2 pmoles of L- cystathionine (except in Strip 6), and were incubated for 30 min at 37”. The enzyme in Strips 5 and 6 was 1 mg of liver cysta- thionine cleavage enzyme with 0.09 ArSSAr unit; in Strip 7, 1.7 mg of Step 3 cleavage enzyme from Neurospora me-d(P162) with 0.08 ArSSAr unit. The reaction mixture of Strip 8 contained 2 pmoles of nn-homoserine, 0.7 pmole of nL-cysteine-%, no carrier cystathionine, and 0.3 mg of liver enzyme; the mixture was treated with performic acid before chromatography in Solvent 1.

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June 1965 C. Delavier-Klutchko and M. Flavin 2545

a net synthesis and not an exchange. It seemed possible that cystathionine synthesis in Neurospora might be catalyzed by the comparable y-cleavage enzyme. However, as shown in Strip 7 of Fig. 3, purified Neurospora enzyme did not incorporate homoserineJ4C into cystathionine under the same conditions in which liver enzyme did.

Many experiments with crude extracts of yeast and Neurospora have also failed to show cystathionine synthesis from homoserine

TABLE VI Nutritional response of Salmonella mutants to succinyl

derivatives of homoserine

Cysteine --f cystathionine + homocysteine ---f methionine

I I I me-A me-C me-E

me-B

Supplement to minimal medium*

nn-Methionine. L-Homocysteine L + n-Allo-cystathionine L-Cysteine 0-Succinyl-DL-liomoserine.. N-Succinyl-nn-homoserine None......................

I Growth response of strain

me-A (15) nwB(l6)

min

0 40 0 40 0 80

2000 100 1400 100 2000 100 3000

min

0 80

0 80 0 80

700 100 700 100

1000 100 1000 100

me-A’t

M Lag 4,

time ‘g - n

min

0 80

0 80

0 180

* Supplements were added in concentrations of from 0.03 to 0.3 pmole per ml. Growth conditions and measurement are described in the text.

t Flasks inoculated from a culture of me-A(15) which had grown on 0-succinylhomoserine after 50-hour incubation.

TABLE VII

Nutritional response of Neurospora mutants to succinyl derivatives of homoserine

Cysteine = cystathionine e homocysteine I I

me-3 me-2 me-5

me-7

Supplement to minimal medium COlICelI- tration

nn-Methionine. L-Homocysteine L -I- n-Allo-cystathionine.. O-Succinyl-DL-homoserine.. iv-Succinyl-nn-homoserine DL-Homoserine

+ Succinic acid. None.......................

)mzole/ml

0.6 0.6 0.6 0.6 0.6 0.6 0.6

.I

Growth response* of strain

me-3 FGSC-

502

175 140 140

0 0 0

0

-

I me-5

‘GSC-140

175 205 125 130 321 205 0 0 0 0 0 0

0 0

me-7 K79

* Dry weight (milligrams per 25 ml) in 72 hours. Other con- ditions are given in the text.

j Delayed onset.

. “ “.” x -- ” ..-.-“. ” ,.... 1 1 3 4 5 ‘~^I’ ..- i

” . . . . . . . .

\ I

/

FIG. 4. Cystathionine synthesis from 0-succinylhomoserine and cysteine catalyzed by extracts of mutant strains of Salmonella. The reaction mixtures were desalted with Dowex 50, oxidized with performic acid, and an aliquot of one-half was then chromato- graphed in Solvent 2. The slower radioactive component cor- responds to the RF of oxidized cystathionine, the faster to cysteic acid. Upper and lower are different experiments. Enzyme prep- arations were dialyzed sonic extracts; apoenzyme 1 is described in the text. The reaction mixtures contained, in 0.5 ml final volume (except where indicated otherwise): 40 pmoles of potas- sium phosphate, pH 7.5; 0.05 pmole of pyridoxal-P; enzyme as indicated; 2.5 pmoles of O-succinyl-nn-homoserine; 2 Kmoles of n-cysteine; 0.2 pmole of nn-cysteine-3% (reduced with hydro- sulfite); incubations were for 30 min at 37” under helium. In the experiment in the lower half of the $gure, reaction Mixture 1 con- tained no enzyme; 2, 0.5 mg from Salmonella me-A; 3, same as

2 + 1OW M hydroxylamine; 4, 0.2 mg of me-A enzyme; 5, 0.15 mg; 6, 0.05 mg; 7, 0.5 mg from me-B; 8, 0.5 mg from me-C. In the ex- periment of the upper half of the $gure, reaction Mixture 1 con- tained me-A apoenzyme 1,0.41 mg; B, same + 1OW M pyridoxal-P; S, same + lo-* M pyridoxal; 4, same preincubated 30 min at 25” with pyridoxal-P; 6, me-A holoenzyme, 0.24 mg; 6, same + 4 pmoles each of ATP and MgC12; 7, same as 5 + nn-cystine-35S, 1 pmole (instead of cysteine) ; 8 same as 7 with gas phase air.

and cysteine, or from derivatives which have been implicated in some cases by earlier indirect results, such as O-phosphohomo- serine, homoserine lactone, S-methylcysteine, or mercaptosuc- cinate.

DISCUSSION

The results show that bakers’ yeast, like Neurospora, can decompose cystathionine by both y and /3 elimination (Reactions

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2546 Synthesis and Cleavage of Cystathionine

TABLE VIII

Vol. 240, No. 6

Elimination and replacement reactions of 0-succinylhomoserine catalyzed by bacterial enzymes

The reaction mixtures all contained, in 1 ml final volume, 100 rmoles of potassium phosphate, pH 7.5, 0.2 ,umole of pyridoxal-P (ex- cept as indicated in apoenzyme incubations), dialyzed sonic or French press extract as indicated, and were incubated at 37” for 30 to

75 min. When cysteine was present, the gas phase was helium. The preparation of apoenzyme is described in the text. After stopping the reactions by addition of 0.1 volume of 1.5 M perchloric acid, the amounts of 0-succinylhomoserine and cysteine consumed were determined as described in the text. The amounts of pyruvate and a-ketobutyrate formed weredetermined with lactic dehydrogenase.

Additions Other additions Substrate consumed Product formed

Experiment 1

None Salmonella me-A Salmonella me-A Salmonella me-B

Experiment 2 E. coli 26/18 E. coli 26/K?

Experiment 3 Salmonella me-A Apoenzyme I

Experiment 4 E. coli 26/H E. coli 26/18

E. coli 26/18 E. coli 26/18 E. coli 26/18

Experiment 5 Salmonella me-A Salmonella me-A

I.- Zysteim Substance a-K&o-

Pyruvate butyrate O-Succinyl- homoserine Cysteine ‘-Succinyl-

mnoserinf

pmoler

5 5

4.2

4.2

5 5

4.2 5.1

Amount

/moles

10,lO

0.2 0.2

5 5

1.3

Protein

w

0

0.2 1.0 1.0

2

2

0.17

0.17

4 4

4 4 4

1.7 1.7

pmozes

5 5 ATP and Mg(CI

3.2 Pyridoxal 3.2 Pyridoxal-P

5 5 5 nn-Homoserine

nn-Homoserine

L-Cystine

pdes pmoles

0.4 0.5

1.3 1.8 2.4 2.5 0* 0

2.6 2.8

0.27 0.13 1.4 1.1

2.7

3.1

1.5 1.9 0.42t

12

0 1.5 0.11 0 0.21 0 0.21 0.13

0 0.32

0.12 1.42

0.58 1.03

+ Sensitivity l/25 of Salmonella me-A.

t Cystine not cysteine.

TABLE IX

Cofactor requirements for y elimination from 0-succinylhomoserine

The reaction conditions and assays were the same as in Table VIII. - Additions Other additions

Wide

0.16

0.16 0

0.06 0.06 0.48 0.10

0.09 0.50

Protein Substance Amount O-Succinyl- homoserine

ATP and MgC12

N-Succinylhomoserine

Pyridoxal Pyridoxal-P

Pyridoxal Pyridoxal-P

/.cmoles

10,5

4

0.4

0.4

0.4 0.4

Experiment 1 E. coli 26/18 E. coli 26118

E. coli 26/18 Experiment 2

Salmonella A apoenzyme 1 Salmonella A apoenzyme 1 Salmonella A apoenzyme 1 Salmonella A apoenzyme 2 Salmonella A apoenzyme 2 Salmonella A apoenzyme 2

2 and 3), whereas E. coli and Salmonella can catalyze only Reac- Extracts of both bacteria were found to catalyze the formation tion 3. Similarly, both fungi can synthesize cystathionine by fl of a compound, chromatographically indistinguishable from replacement (Reaction I), whereas neither of the bacteria can do cystathionine, from cysteine and O-succinyl-nn-homoserine so. It is very likely that pyridoxal-P is required for all these (Reaction 4). Contrary to previous reports (20) ATP was not, reactions; the requirement has been shown for Reaction 1 in liver and pyridoxal-P was, required. The rate of this reaction was un- (2), React,ion 2 in liver (1) and Neurospora (8), and Reaction 3 in usually high, for a biosynthetic reaction, in the bacterial extracts; E. coli (13).3 it could not be detected in either of the fungi. In the absence of

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June 1965 C. Delavie~-Klutchko and M. Flavin 2547

cysteine, both bacterial and Neurospora extracts decomposed 0-succinylhomoserine by Reaction 5. Reactions 4 and 5 are catalyzed by the same enzyme in bacteria, since both are missing from the same methionine auxotroph, Salmonella me-B(16).

However, in Neurospora Reaction 5 was catalyzed by an enzyme whose main physiological role appears to be to decompose cysta- thionine by Reaction 2.

The enzyme catalyzing Reactions 4 and 5 in the bacteria must be required for methionine synthesis since it is missing from a methionine auxotroph. However, it is not equally sure from these results that 0-succinylhomoserine is a natural intermediate in methionine biosynthesis. The enzymatic synthesis of this compound has not yet been fully characterized, although avail- able evidence indicates that it, arises from a reaction between succinyl CoA and homoserine (20) catalyzed by an enzyme missing from Salmonella me-A mutants. The inability of O- succinylhomoserine to support the growth of appropriate Sal- monella methionine auxotrophs raises the possibility that it might be closely related but not identical to the natural intermediate reported by Rowbury and Woods (20), and makes desirable an unequivocal demonstration of the structure of the reaction prod- uct. Recently, as will be reported elsewhere (16), the latter has been isolated in crystalline form and proved to be n-cystathionine. The nutritional inertness of 0-succinylhomoserine may be due to impermeability, or to an extracellular or membrane est,erase.

The rate of Reaction 4 catalyzed by the bacterial enzyme is about 10 times greater than that of Reaction 5. This rate dif- ference may not be enough to explain the apparently complete lack of a-ketobutyrate formation in the presence of cysteine. Inhibition of pyridoxal-P reactions due to thiazolidine formation by cysteine is usually incomplete under these conditions, as il- lustrated by the partial inhibition of a+ketobutyrate formation from homoserine (Table VIII). In any case, since cysteine is a substrate for t’his pyridoxalP enzyme in Reaction 4, the co- enzyme may be shielded from thiazolidine formation with cys- teine. It is therefore possible that cysteine inhibits Reaction 5 by changing the catalytic properties and conformation of the enzyme.

The conclusion that transsulfuration via cystathionine is reversible in Neurospora and proceeds only from cysteine + homocysteine in E. coli accords with that reached by some earlier indirect studies (18, 19, 23) and conflicts with others (24). With the aid of more sensitive methods for detecting the liberation of small amounts of alternative products from cystathionine, these results also confirm the conclusions reached from enzymatic studies of E. coli in the laboratory of the late D. D. Woods (14).

E. coli evidently can slowly transfer sulfur from methionine to cysteine by some other path, probably by way of inorganic sulfur compounds, since we have found that strains used in this work can grow with methionine as the sole source of sulfur.3

In the forward direction of methionine biosynthesis (cysteine -+ homocysteine) the reactions mediating the transfer of sulfur from cysteine to cystathionine have not yet been discovered in Neuro- spora and yeast. The negative results with O-succinylhomo- serine do not prove that it is not an intermediate in the fungi. The principle indication of some difference from the bacterial pathway remains the fact that three, rather than two, genetic loci appear to be involved. In the biosynthesis of lysine, another member of the “aspartate family” of amino acids, a succinyl- ated intermediate is also involved in E. coli, but not in the higher fungi (25). There is no thermodynamic objection to the syn-

O-SUCC- HSW

PYr

“HS.3” aKB

pur

Escherichia coli

Salmonella

Neurosporo Yeast Mammals

FIG. 5. Patterns of transsulfuration. 0-Succ-Hser, O-succinyl- homoserine; Hser, homoserine; aKB, a-Ketobutyrate; Hcys, homocysteine; and Pyr, pyruvate.

thesis of cystathionine directly from cysteine and homoserine itself (17); the formation of a thioether from an alcohol and a mercaptan is strongly exergonic (26). Although an ester is a better leaving group than an unprotonated hydroxyl, there is also no kinetic requirement for succinylation of homoserine. Acceleration by the enzyme is of over-riding importance, as il- lustrated by the rates of reaction of various substrates for cysta- thionine cleavage enzymes (8, 22). However, synthesis of cystathionine directly from homoserine could also not be shown with extracts of Neurospora or yeast.

Present knowledge of biological patterns of transsulfuration is summarized in Fig. 5. The value of this very limited survey of organisms is to suggest that the distribution of the various en- zymes is not capricious; i.e. the enzymes catalyzing Reactions 1 and 2 are both present in two higher fungi, and both absent from the two closely related bacteria. Some problems which might be illuminated by a wider survey are mentioned below.

What is the utility of reverse transsulfuration (homocysteine + cysteine) in mammals and higher fungi, and why have the bac- terial species dispensed with this process? The wide range of substrates decomposed by the enzymes cat’alyzing Reactions 1 and 2 (2, 8, 22) raises the question, at first, of whether the prin- ciple function of the enzymes is, in fact, to transfer sulfur from methionine to cysteine. Available evidence, particularly dietary effects on enzyme levels in liver (27-29), suggests an affirmative answer. The question then becomes one of the utility of sulfur transfer from methionine to cysteine. The only mutants known to be blocked in Reactions 1 and 2 are in man. In these cases the single absence of either enzyme is clearly deleterious to the whole organism (30, 31), as are many other imbalances in amino acid metabolism.

In Neurospora and yeast the most plausible cellular utility of reverse transsulfuration might be to conserve sulfur efficiently during a phase of the complex life cycle of these sporulating fungi when nutritional deprivation requires utilization of endogenous materials for the synthesis of new structures. During early sporulation of Bacillus species the cell content of cystine increases 5fold with concomitant decrease of methionine (32). It is not clear whether this process occurs during nutritional deprivation, but in any case exogenous cystine (10e4 M) inhibits sporulation (32). It would be informative to determine the pattern of trans- sulfuration in these sporulating bacteria, and to examine enzyme levels throughout their life cycle.

Alternatively, reverse transsulfuration might serve some regulatory function in the multichromosomal fungi. For the methionine synthetic pathway in bacteria, the familiar pattern

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2,548 Synthesis and Cleavage of Cystathionine

Fossil organisms

Methionine heterotroph Fungal

Methionine O-Succinyl- Methionine C pathway

heterotroph + homoserine + heterotroph + (reverse trans- + (reverse A pathway B sulfuration) transsulfuration)

4 I 1 E. coli Mammals Yeast Salmonella Neurospora

Modern organisms

SCHEME 1

Vol. 240, So. 6

of regulation by feedback and repression has been reported (14, 15). Reverse transsulfuration might provide an alternate or supplemental control of the cellular methionine concentration in yeast and Neurospora. Parenthetically, the ability of the same enzyme to catalyze Reactions 4 and 5 suggests a possible supple- mental control of the bacterial synthesis of methionine. De- ficiency of methionine consequent to deficiency of cysteine would result in uncont’rolled over-production of 0-succinylhomoserine. While the latter could equally well be removed by a nonspecific esterase, the total inhibition of Reaction 5 by cysteine would provide more effective regulation and conservation of energy. However, the conclusion that the dual capability of the bacterial enzyme does have regulatory utility is subject to a reservation. In the case of Reaction 5, its existence may not compel the con- clusion that it) confers a selective advantage, since the y elimina- tion could be “uncatalyzed.” The actual intermediate, formed from 0-succinylhomoserine, which reacts with cysteine, may be spontaneously unstable (7) in t,he absence of the latler. In the only analogous enzymatic reaction, the formation of threonine from 0-phosphohomoserine, a significant alternate formation of a-ketobutyrate has not been observed.* But in this case the attacking group is water and it can not be excluded from the reaction mixture.

The pat’tern shown in Fig. 5 raises several questions concerning the evolutionary origin of transsulfurat’ion, which can in part be formulated in analogy to studies of the evolution of lysine biosyn- thesis (25). Assuming that’ the fungal pathway from cysteine to cystathionine differs from the bacterial pathway, it could be inferred that these originated at different times (25) and were separated by an ancestral methionine heterotroph B (Scheme 1). A special feature in this case is that the separately evolving pathways would contain Reaction 3 in common. The enzymes catalyzing Reaction 3 might then be expected to fall into two sharply differentiated groups. These might be distinguishable by their functional properties alone, in view of the wide range of substrates decomposed by the cystathionine cleavage enzymes so far purified (8, 22).3 The most striking difference shown here between the bacterial and Neurospora cystathionine P-cleavage enzymes is in their susceptibility to inhibition by EM and ArSSAr. It will be informative to confirm the preliminary indications that the yeast enzyme (Table I) resembles that from Neurospora in this respect.

The origin of reverse transsulfuration presents an additional problem. The simpler supposition would be that this pathway originated only once, and since, among the few organisms so far studied, none have been observed which contain the fungal pathway without the back reactions, that its origin (“methionine heterotroph C”) preceded that of the fungal pathway. A com-

parison of the mammalian and Neurospora enzymes catalyzing Reactions 1 and 2 might again be informative, and in the case of the cystathionine y-cleavage enzymes (8,22) has already revealed many functional similarities.

SUMMARY

The pyridoxal phosphate-dependent reactions mediating the transfer of sulfur between cysteine and homocysteine (trans- sulfuration) have been investigated in several microorganisms. The cystathionine-mediated pathway has been shown to be reversible in two species of higher fungi, and irreversible from cysteine to homocysteine in two species of bacteria. Yeast and Neurospora cleave cystathionine both to cysteine + a-ketobuty- rate (y-cleavage), and to homocysteine + pyruvate (P-cleavage). Escherichia coli and Salmonella contain only a p-cleavage enzyme. The bacterial cleavage enzymes differ from the fungal P-cleavage enzymes in their insensitivity bo inhibition by maleimides and aromatic disulfides. They are absent from E. coli 26/18 and Salmonella me-C(30) grown at 37”; the Salmonella mutant forms some active enzyme at 25”.

Both Neurospora and yeast can synthesize cystathionine from serine and homocysteine (0 synthesis) ; neither bacterium can do so. The Neurospora p synt,hetase is distinct from the /3- or the y-cleavage enzyme.

The succinic amide and ester of homoserine were synthesized. Neither supports the growth of methionine auxotrophs of Neuro- spora or Salmonella. However, bacterial extracts catalyze a rapid synthesis of cystathionine from cysteine and the ester. The y synthetase could not be detected in Neurospora, yeast, or Salmonella me-B(16). In t’he absence of cysteine, the same bacterial enzyme decomposes 0-succinylhomoserine to cu-keto- butyrate; this react,ion is completely inhibited by cysteine. Both reactions require pyridoxal-P.

The pathway of cystathionine synthesis from cysteine has not been established in t,he higher fungi. Neurospora cystathionine y-cleavage enzyme does not cat’alyze y-synthesis directly from homoserine and cysteine under the same conditions where the comparable liver enzyme slowly does so.

These patterns of enzyme distribution are discussed in relation to the evolutionary origins and present functions of transsulfura- tion.

Acknowledgments-For the mutant strains used in this work, we are indebted to Drs. N. E. Murray, M. Demerec, G. N. Cohen, B. D. Davis, A. L. Taylor, and J. B. Loerch. We also thank Dr. D. M. Greenberg for a lyophilized sample of crystalline liver cystathionine cleavage enzyme.

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Colette Delavier-Klutchko and Martin FlavinEnzymatic Synthesis and Cleavage of Cystathionine in Fungi and Bacteria

1965, 240:2537-2549.J. Biol. Chem. 

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