Mechanism of action of cystathionine synthase

ARCHIVES OF BIOCHEMISTRY Vol. 213, No. 2, February,

AND BIOPHYSICS pp. 695-70’7, 1982

Mechanism ERIC

of Action of Cystathionine BORCSOK’

Graduate Department of Biochemistry,

ROBERT

AND

Bran&is

Received

July

University,

Synthase’

H. ABELES Waltham, Massachusetts 02254

29, 1981

Cystathionine synthase catalyzes the formation of cystathionine from serine, as well as @-chloroalanine, and homocysteine. The stereochemistry of the replacement of the p-OH group of serine by homocysteine was established by converting cystathionine, derived from 2,S’, 3R-[3-3H]serine, to cysteine with y-cystathionase. This conversion does not alter the configuration at the /3 carbon of cystathionine. With previously used procedures (H. G. Floss, E. Schleicher, and R. Potts (1976) J. Biol Chem 251, 5478-5483) it was determined that the cysteine so obtained was 2R, 3R-[3-3H)cysteine, hence the displacement of the OH group of serine by homocysteine proceeds with retention of configuration. The kinetics of the replacement reaction show an intersecting line pattern. This result, together with the observation that, in the absence of homocysteine, little, if any catalytic conversion of @-chloroalanine to serine occurs, indicates that the /3 substituent is not released from the enzyme prior to binding of homocysteine. In the absence of homocysteine, the enzyme catalyzes exchange of tritium from [ol-3H$serine and P-[cY-3H]chloroalanine with solvent protons. At pH 7.8, the rate of this exchange reaction is nearly equal to V of cystathionine formation. The rate of pyruvate formation from serine or fi-chloroalanine in the absence of homocysteine is maximally 2% that of the tritium exchange rates. These results show that, in the absence of homocysteine, cystathionine synthase catalyzes a-proton abstraction more rapidly than elimination of the @-substituent. Several mechanisms are proposed by which elimination of the B substituent from the substrate-derived carbanion is prevented in the absence of homocysteine. Cystathionine synthase is irreversibly inactivated by 30 mM 2-amino-4-chlorobutyric acid with t”’ = 31 min.

Cystathionine synthase (EC 4.2.1.22), a pyridoxal phosphate-requiring enzyme (l), catalyzes the replacement of the P-OH group of serine by homocysteine to form cystathionine:

This enzyme plays a central role in the degradative metabolism of homocysteine in higher animals. Its absence in some humans results in homocysteinuria (2), a disease with a number of serious pathological consequences. We tested &/3,/3-trifluoroalanine as a potential suicide inactivator for this enzyme, and hoped to use it to generate an animal model for homocysteinuria. However, P&3$-trifluoroalanine did not inactivate cystathionine synthase but acted as a competitive inhibitor. This was surprising since &/?,p-trifluoroalanine inactivates other enzymes which catalyze P-replacement and P-elimination reactions: tryptophan synthase, tryptophanase, ,&cystathionase, y-cystathionase (3). Enzymes that catalyze ,&replacement and P-elimination reactions are generally

1 This research was supported in part by a grant from the National Institutes of Health (GM 1263318) to R. H. Abeles. E.B. was supported from a National Institutes of Health Training Grant (GM 00212). ‘Present address: Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass. 02139.

695

0003-9861/82/020695-13$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

696

BORCSOK

AND

believed to operate through similar mechanisms (Eq. [Z] (4)). It is, therefore, surprising that &&@trifluoroalanine does not inactivate cystathionine synthase. This failure to inactivate could simply be due

ABELES

to nonproductive binding. On the other hand it is possible that the mechanism of action of cystathionine synthase, and perhaps that of other enzymes that catalzye P-replacement reactions, differs from the mechanism of Eq. [2].

PY

(Py = pyridoxyl

phosphate,

a = @ elimination,

Possibly, /3-cystathionine synthase does not catalzye a-proton abstraction from serine or elimination of the /3 substituent until homocysteine is present. Another possibility is that the reaction could proceed through direct displacement as shown in Eq. [3].

-

i&coo---+ ‘Z bY

r12-y”-coO+NH3

According to this mechanism, the p substituent is activated toward displacement by formation of an imine at the a-carbon. A similar mechanism has also been considered by Braunstein (5). If a displacement mechanism is involved, it is expected that replacement of the /3 substituent proceeds with inversion of configuration. For P-replacement reactions investigated so far, this is not the case (6-9). We now report the results of an investigation of the mechanism of action of cystathionine synthase in which we examined the structural modification of serine catalyzed by cystathionine synthase in the absence of ho-

b = p replacement).

mocysteine, as well as the stereochemistry of the replacement reaction. MATERIALS

Putification

AND

METHODS

of Cystathimine

Synthase

Ten rats were decapitated, the livers were removed and put on ice. All subsequent steps were carried out at 4°C unless otherwise noted. The livers were then washed with 0.1 M potassium phosphate, pH 7.2, and homogenized with 3 vol of 50 mM potassium phosphate, pH 7.2, 0.15 M KCl, 1 mM EDTA in a Waring blender for 1 min. The homogenate was centrifuged at 10,OOOg for 20 min. To the supernatant fluid, solid ammonium sulfate (14 g/100 ml, 25% saturation) was added in five portions with stirring. The pH was maintained at 7.2 with 10% ammonium hydroxide. One hour after the initial addition, the precipitate was removed by centrifugation for 20 min at 10,OOOg. The supernatant fluid was brought to 40% saturation with the addition of 9 g (NH&./100 ml, added in three portions over 1 h. The precipitate was collected by centrifugation for 40 min at 10,OOOg and the pellet was suspended in 50 ml of 10 mM potassium phosphate, pH 7.8, 1 mM mercaptoethanol, and 1 mM EDTA (Buffer A). The enzyme was dialyzed against Buffer A. The dialyzed fraction was applied to a 3.4 X 20-cm DEAE column that had been equilibrated with Buffer A. The column ‘was washed with 330 ml of Buffer A, then with 330 ml 50 mM potassium phosphate, pH 7.8, 1 mM mercaptoethanol, and 1 mM EDTA, followed by 500 ml of 100 mM potassium phosphate, pH 7.8, 1 mM mercaptoethanol, and 1 mM EDTA. The enzyme was eluted with the last buffer.

RAT

CYSTATHIONINE

SYNTHASE

The active fractions were pooled and dialyzed for 24 h against three changes of 10 vol of 3 mM potassium phosphate, pH 6.80, 1 mM mercaptoethanol, and 0.1 mM pyridoxal 5’-phosphate. Hydroxyapatite was prepared by the method of G. Bernardi (10). Fine particles were removed by suspension in 3 mM potassium phosphate, pH 6.8,l mM mercaptoethanol, and 0.1 mM pyridoxal5’-phosphate. A 3.4 X 6-cm column was prepared. This column was equilibrated with the same buffer and the protein was applied at a concentration of 3 mg/ml, with a flow rate of 1 ml/min. The column was washed with the equilibration buffer until the ODzso of the effluent was below 0.2. A linear gradient consisting of 200 ml of 3 mM potassium phosphate, pH 6.8,l mM mercaptoethanol, and 0.1 mM pyridoxal 5’-phosphate in the first flask and 200 ml of 0.3 M potassium phosphate, pH 6.8, 1 mM mercaptoethanol, and 0.1 mM pyridoxal 5’-phosphate in the second flask was applied. Column effluent fractions with a specific activity greater than 0.4 u/mg were pooled. Solid ammonium sulfate was added to saturation and the pH adjusted to 7.4 with 10% ammonium hydroxide. After stirring 1 h the precipitate was removed by centrifugation at 10,OOOg for 20 min. The pellet was resuspended in Buffer A and dialyzed against Buffer A overnight. A 1.6 X 20-cm Whatman DE-52 column was equilibrated with 10 mM potassium phosphate, pH 7.8, 3 M urea, 1 mM EDTA, and 1 mM mercaptoethanol (Buffer B). The enzyme was diluted with an equal volume of Buffer B containing 6M urea and 1 mM EDTA, and was applied to the column (flow rate 1 ml/l.5 min). A linear gradient consisting of 150 ml of Buffer B in the first flask and 150 ml of 0.1 M potassium phosphate, pH 7.8, 3 M urea. 1 mM EDTA, and 1 mM mercaptoethanol in the second flask was applied. Fractions with a specific activity greater than 3.0 u/mg were pooled, diluted with 10 vol 4°C distilled water, and applied to a 0.7 X 3-cm DEAE column equilibrated with Buffer A. This was followed by 3 ml of Buffer A, then with 3 ml of 0.1 M potassium phosphate, pH 7.8, 1 mM EDTA, and 1 mM mercaptoethanol. The enzyme was eluted with the last buffer and could be further concentrated by ultrafiltration (Amicon).

Sephadex

G-150 Gel Filtration

Several enzyme preparations taken through the procedure described above were dialyzed overnight against 50 vol of 10 mM potassium phosphate, pH 6.8, 5 mM EDTA, 1 M urea, and 0.3 M sodium chloride. The enzyme was then applied to a 1 X 60-cm Sephadex G-150 column equilibrated with 10 mM potassium phosphate, pH 7.8, 1 mM EDTA, 1 mM mercaptoethanol, and 0.2 M sodium chloride, and eluted with the same buffer. Active fractions of specific activity greater than 4.0 u/mg were pooled and concentrated

by Amicon ultrafiltration A typical purification

Enzyme

697

MECHANISM with protocol

a PM 10 membrane. is shown in Table I.

Assay

Cystathionine synthase was assayed by a modification of published procedures (11,12). Cystathionine synthase was incubated in a total volume of 50 ~1 with 8 mM L-[U-14C]serine, 64 mM DL-homocysteine, 270 mM Tris . HCl, pH 8.60, 1 mM EDTA at 37°C for 30 min. Five microliters of the mixture was then withdrawn and spotted onto a cellulose TLC3 plate previously spotted with 1 ~1 of 20% trichloroacetic acid and 1 ~1 of 50 mM cystathionine. The TLC plate was then developed in N-butanol/acetic acid/water (12/5/3, v/v/v). An extra lane spotted with cystathionine was also present and the cystathionine was located with a ninhydrin spray (0.5% in acetone). The region corresponding to cystathionine was sliced out and counted in 4 ml of ACS counting solution. One unit of enzyme is defined as the amount of enzyme that catalyzes the formation of 1 rmol of cystathionine per minute at 37°C. Enzyme activity was also measured by determining the amount of ‘Hz0 produced when [&H]serine was added to the enzyme. The reaction mixture contained DL-[cu-aH]serine 10 mM, DL-homocysteine 64 mM, EDTA 4 mM, Tris . HCl, pH 8.6, 350 mM, or potassium phosphate buffer 350 mM, pH 7.8, in a total volume of 250 ~1. After 15 min at 37°C the reaction was stopped by addition of 100 ~120% TCA, and was then applied to a Dowex 50 H’ (200-400 mesh) column, 0.7 x 30 cm. The column was eluted with 2 ml of water and the column effluent was added to 10 ml of ACS (New England Nuclear Corp.) scintillation fluid for determination of radioactivity. Maximal utilization of serine in this assay was 10%.

Determination of Products Serine and /3-Cl-Alanine

Derived

from

Cystathionine synthase (sp act 1.04 u/mg) was incubated with 2 mM L-[U-14C]serine, 280 mM Tris. HCl, pH 8.60, and 2.8 mM EDTA in a total volume of 250 ~1. The reactions were allowed to proceed 6 h at 37°C and terminated by addition of 100 pl of 20% trichloroacetic acid and 10 ~1 of 0.1 M sodium pyruvate. The reaction mixture was applied to 0.7 X 3-cm Dowex 50 (H+ form) columns and eluted with 10 ml water. Fractions (2 ml) were collected; the amount of radioactive material in each fraction was determined. Pyruvate was found to elute in the first fraction using a lactate dehydrogenase assay (13). Fur-

’ Abbreviations used: TLC, thin-layer raphy; TCA, trichloroacetic acid; SDS, decyl sulfate.

chromatogsodium do-

698

BORCSOK

AND

ther characterization of pyruvate is described below. For identification of the products derived from @Cl-alanine the following conditions were used: 280 mM potassium phosphate, pH 7.80, 12 mM L-[Ui4C]chloroalanine, 100 ~1 of cystathionine synthase (sp act 1.04 u/mg) in a total volume of 250 pl. After incubation at 37°C for 400 min, 2 nmol sodium pyruvate and 5 pmol serine were added and the mixture was applied to a 0.7 X 3-cm Dowex 50 (H+ form) column. A blank without enzyme was also incubated. The columns were eluted with 2 ml of water and the effluent assayed for pyruvate with lactate dehydrogenase (13). To 1 ml of effluent, 3 mg of sodium pyruvate was added and the pH was adjusted to 8.0 with 1 M sodium hydroxide and then lyophilized. The residue was dissolved in 0.20 ml of 0.5 M HzS04, then mixed with 0.5 g silicic acid, previously dried at 100°C overnight. Pyruvate was isolated and purified by chromatography on a silicic acid column (1 X 10 cm) (14). Pyruvate was detected spectrophotometrically (13). The specific radioactivity throughout the peak was constant. An aliquot of the pyruvate from the silicic acid column was converted to the 2,4-dinitrophenylhydrazone. The specific activity of the 2,4-dinitrophenylhydrazone was determined by dissolving it in ethanol. The radioactivity present in an aliquot was determined and the concentration of the 2,4-dinitrophenylhydrazone was determined spectrophoChloratometrically (cQw nm = 2.18 X 10e3 M-l cm-‘). lanine and serine were separated by transferring the ion-exchange resin from the small column to a 1 x 15-cm Dowex 50 (H+ form) column equilibrated with 0.5 M hydrochloric acid. The column was eluted with 0.5 M hydrochloric acid; serine eluted first, then chloroalanine. Amino acids were detected with the ninhydrin assay (15). The specific activity across the serine peak was constant and the recovery of serine was quantitative.

Synthetic Procedures Homoqpteine. Homocysteine was synthesized by the procedure of M. Suda et al. (11). DL-[a-3H]Serine was synthesized by the procedure of Miles and McPhie (14). DL-fla-SH]chkwoakwzine

and DLflU-*4CJchloroala-

nine. DL-[a-aH]Serine methyl ester * HCl was prepared from DL-[a-3H]serine (16). The methyl ester was then converted to DL-@-[&H]chloroalanine (17). This product was isolated by preparative paper chromatography on Whatman 3MM paper (prewashed by downward elution with water). The chromatogram was developed in an ascending fashion with N-butanol/water/acetic acid (121513). A strip was cut off from each side of the paper, to determine the location of radioactive materials. The radioactive band corresponding to chloroalanine was cut out, eluted with water, and lyophilized to yield 0.0127 g (6.9 X 10e5

ABELES

mol) DL-[cY-3H]chloroalanine (91% from DL{asH]serine methyl ester hydrochloride). It was found radiochemically pure by cellulose TLC in N-butanob pyridinelwater (l/l/l) and N-butanol/acetic acid/ water (121315). L-[U-i4C]Chloroalanine was synthesized as described above except that L-[U-i4C]serine was used instead of DL-[a-sH]serine. Radiochemical purity was established by chromatography on Dowex 50 (H+ form) and paper chromatography in N-butanol/acetic acid/water (12/3/5). 2-Ami?w+&knw&&enoic acid. 2 Amino-3-chloro3-butenoic acid was synthesized as follows: 408 mg (3 mmol) of 2-hydroxy-3-chloro-3-butenoic acid (recrystallized from chloroform) in 2 ml anhydrous diethyl ether was added to 0.18 ml (2 mmol) phosphorous tribromide and 0.020 ml pyridine (0.25 mmol) in three portions over 60 min. The mixture was stirred an additional 20 min, 5 ml water was then added, and stirring continued for 1 h. The organic layer was concentrated to dryness in rucuo and the flask placed on ice. Concentrated NHIOH (9 ml) cooled to 5°C was added to the residue and the mixture allowed to come to room temperature. After 25 h, the mixture was concentrated in ‘uacuo. Water (1 ml) was added, and the pH was adjusted to 8 with 1 M HCl. The solvent was then removed in 2)acr~o and the oil was triturated with acetone. The product slowly crystallized. The 90-MHz NMR spectrum in DzO gave the following assignments: C-2H 4.75 (s), C-4H (tram) 5.65 (d), C-4H (ci.s) 5.75 (d). Elemental analysis for chlorine gave 26.22% Cl found, 26.20% calculated.

2-Amiw&chlorobut~ric acid. 2-Amino-4-chlorobutyric acid was made by a modification of the procedure of Valle (18). a-Aminobutyric acid, 1 g (9.4 mmol) dissolved in 5.3 ml concentrated hydrochloric acid, was chlorinated at 70°C by bubbling chlorine gas through the reaction mixture and adding 1.76 g (10.7 mmol)azobis(propionitrile) in 12 portions over a 6-h period. The solvent was removed by rotary evaporation. The residue was applied to a Dowex 50 (H+ form) column (1.5 X 40 cm) and eluted at 1 ml/ min with a linear gradient consisting of 500 ml HZ0 in the first flask and 500 ml 3 M hydrochloric acid in the second flask. These fractions containing 2-aminoI-chlorobutyric acid were pooled and concentrated in a rotary evaporator to give a white solid in quantitative yield, mp 155-156”C, lit. 154-156°C.

Analytical

Procedures

High-voltage paper electrophoresis was carried out in Savant HVE-8036 tanks with Whatman 3MM paper 57 cm long. The paper was prewashed by downward elution with water. The pH 1.9 buffer was 2% formic acid (v/v), 8% acetic acid (v/v) in water. Electrophoresis at this pH was carried out at 4000

RAT CYSTATHIONINE V for 30 min; the origin was 15 cm from the positive pole. Radioactivity on chromatograms was located with a tracer lab 4a strip scanner or by cutting the chromatogram into l-cm-wide slices and counting each slice in 4 ml of ACS (Amersham). Radioactivity was determined with a Beckman LS106 C liquid scintillation counter. ACS scintillation fluid was purchased from Amersham. Counting efficiencies were determined by adding known amounts of [‘“Cl- or pH]toluene to each sample. SDS-gel electrophoresis was carried out by the procedure of Weber and Osborn (19). Protein was stained with 0.25% Coomassie Blue 250 in methanol/ acetic acid/water (5/l/5, v/v/v) overnight and destained in 10% methanol, 10% acetic acid at 40°C. Amino acids were detected on chromatograms by spraying the chromatograms with 0.5% ninhydrin in acetone and placing the sheets in a 100°C oven for 1 min.

Determination of the Stereochemistry of the Cystathionine Synthuse Reaction For the determination of the stereochemistry the reaction sequence shown in Scheme 1 was used. Reaction I. 3R-[3-3H]- or 3S-[33H]serine, 300 nmol (5 pCi 3H), DL-homocysteine, 1.6 rmol, Tris * HCI, 40 pmol, pH 8.6, cystathionine synthase (0.4 IU/mg), 0.26 IU, were incubated at 37°C in a total volume of 147 ~1 for 27 min. The reaction mixture was then applied to a 0.7 X 4-cm Dowex 50 (H+ form) column and eluted with 3 ml water, then 20 ml of 0.4 M hydrochloric acid, then 5 ml of 1 M pyridine. The pyridine effluent, which contained [3-3H]cystathionine, was concentrated by rotary evaporation to dryness and dried in vacua over P205 overnight. The yield of cystathionine was 95% based on serine. Reaction 11, III. To [3-3H]cystathionine was added 50 nmol pyridoxal phosphate, 840 nmol dithiothreitol, 7 pmol EDTA, 105 ~1 of 1 M potassium phosphate, pH 6.8, 800 nmol NADH, and 126 units lactate dehydrogenase in a total volume of 1.3 ml. The reaction flask was sealed with a serum cap and purged with argon for 1 h. y-Cystathionase, 0.8 unit (sp act 300 u/mg) in 100 ~1 was added with a syringe and the reaction allowed to proceed 50 min at 37°C. Methyl pnitrobenzene sulfonate (9 mg) was then added as a finely ground powder (7) and the flask was purged with argon for 10 min. After the reaction had proceeded for 24 h at 37°C the mixture was applied to a 1 X 16-cm Dowex 50 (H+ form) column and a linear gradient of 100 ml of distilled water in the first flask and 100 ml of 3 M hydrochloric acid in the second flask was applied. Fractions (2.5 ml) were collected every 3.5 min and S-methylcysteine was eluted in fractions 57 to 73 as judged by cellulose TLC in N-

SYNTHASE

MECHANISM

699

butanol/acetic acid/water (12/3/5). These fractions were pooled and concentrated in ‘umo to an oil. The yield based on cystathionine was 83%. S-Methylcysteine so obtained was more than 97% pure as judged by high-voltage paper electrophoresis at pH 8.9, using 1% ammonium carbonate as buffer. Reaction IV. To 9[3-3H]methyl-L-cysteine was added 0.5 mg (3.7 pmol) nonisotopic S-methyl-L-cysteine and 200 ~1 boiling glacial acetic acid. Acetic anhydride (25 ~1) was then added and the mixture was boiled for 2 min and then allowed to cool to room temperature. The solvent was removed in a vacuum desiccator over potassium hydroxide to give N-acetyl-S-[3-3H]methyl-DL-cysteine in quantitative yield; mp 150-151°C lit. 151-152°C (7). The N-acetyl-S-[3-3H]methyl-DL-cysteine was dissolved in 0.5 ml 60% formic acid and 15 mg cyanogen bromide was added. The reaction was allowed to proceed at room temperature for 72 h. Solvent was then removed by rotary evaporation, 3 mg L-serine was added and the mixture was applied to a 1 X 15-cm Dowex 50 (H’ form) column. The column was eluted with 75 ml of 0.5 M hydrochloric acid followed by 200 ml of 3 M hydrochloric acid. Fractions (2.5 ml) were collected every 4 min. The serine was eluted in fractions 37 to 45, S-methylcysteine in fractions 73 to 95. Fractions containing serine were concentrated by rotary evaporation and L-[U-i4C]serine added. Aliquots were removed for the determination of the 3H/ ‘*C ratio. Reaction V. To 28.6 wmol DL-~H, i4C]serine was added 30 pmol indole, 2 rmol pyridoxal5’-phosphate, 1.25 ml of 1 M potassium phosphate, pH 7.2, and 6 units of tryptophan synthetase in a total volume of 10.0 ml. The reaction was maintained for 200 min at 37°C then concentrated by rotary evaporation to an oil. The residue was dissolved in 200 ~1 water, applied to two 20 X 20-cm cellulose TLC plates, and developed in N-butanol/acetic acid/water (12/3/5). The band corresponding to tryptophan (RI 0.50, visualized by uv lights) was scraped off the TLC plate, placed in a Pasteur pipet plugged with glass wool, and eluted with 10 ml of boiling water. The effluent was concentrated in wacuo to a white solid. The yield of tryptophan was 85% based on serine. Tryptophan was radiochemically pure as judged by cellulose TLC in methanol/pyridine/water @O/5/20, v/v/v), and Nbutanol/formic acid/water (77/10/3, v/v/v). An aliquot was withdrawn for determination of the 3H/14C ratio. Conversion of S-[3-SHjMethyl-.Kysteine to L-Tryp tophan. To 25 pmol 9[3-3H]methyl-L-cysteine was added 30 pmol indole, 2 pmol of pyridoxal 5’-phosphate, 1.25 ml of 1 M potassium phosphate, pH 7.2, 20 units of tryptophan synthetase. The reaction mixture was sterilized by filtration through an autoclaved Millipore filter (type HA, 0.45-pm pore diameter). The reaction was then allowed to proceed

700

BORCSOK

AND

for 12 h at 3’7°C and then applied to a 0.7 X5-cm Dowex 50 (H+ form) column. The S-methylcysteine was eluted with 20 ml of 3 M hydrochloric acid. L-[3-3H]Tryptophan was eluted with 10 ml of 2M ammonium hydroxide and lyophilized. L-[U-'4C]Tryptophan was added and aliquots were withdrawn to determine the ‘H/i% ratio. [‘H/%]Tryptophan is radiochemically pure as judged by cellulose TLC in the systems described above. Reaction VI. The tryptophan samples were converted to indolmycin by incubation with 25 ml shake cultures of Streptmyces gv%wu.s ATCC 12648 in a sporulation broth consisting of l.Og yeast extract, 1.9g beef extract, 2.9g tryptose, 1 mg FeS04, log glucose, and 3 liters HzO. The fermentation as well as the isolation of indolmycin were carried out as described earlier (20). The indolmycin was assayed for its 3H/‘4C ratio.

ABELES

RESULTS

Enzyme Purification

The results of the enzyme purification are summarized in Table I. The enzyme was purified 2250-fold. Previously this enzyme had been purified 1212-fold (1). The major difference between this purification procedure and the previously published procedure is chromatography on DEAEcellulose in 3.5 M urea. The purified enzyme shows a single band on SDS-acrylamide electrophoresis. The subunit molecular weight is 53,000. To obtain additional evidence that the band observed on electrophoresis is cystathionine synthase, a few crystals of rH]NaBH, were added to the enzyme prior to electrophoresis. Presumably rH]NaBH, reduces the Schiff base between pyridoxal phosphate and the lysyl c-NH2 group and would therefore introduce 3H into the enzyme. The inactivation and reduction of cystathionine synthase by NaBH, has been reported (1). Upon electrophoresis of the reduced enzyme, essentially all of the radioactivity was found with the protein band. A molecular weight of 112,000 (1) has been determined by gel chromatography. Therefore, the native enzyme appears to be a dimer of identical or nearly identical subunits. The enzyme is extremely hydrophobic. Thus, the enzyme binds tightly to bromophenol blue (dye used in electrophoresis), to alkyl Sepharose columns (straight chain C&,,), and

Materials Pyridoxal, amino acids, trizma base, Dowex resins, and Sephadex gels were purchased from Sigma Chemical Company. 3Hz0 (1 Ci/ml), L[U-‘%]serine and L-[3-14C]tryptophan were obtained from New England Nuclear. &/3&Trifluoroalanine and 2-hydroxy-3-chloro-3-butenoic acid were a gift of Dr. R. B. Silverman. Phosphorous pentachloride and DLhomocysteine thiolactone * HCl were purchased from Aldrich Chemical Company. All other reagents were obtained from Fisher Chemical Company. Growth media were Difco Products. S griseus ATCC 12648 was obtained from the American Type Culture Collection and was maintained on plates of Emerson agar at 24°C. y-Cystathionase was kindly provided by Dr. Barbara Lapinskas and Clare Fearon. E. coli tryptophan synthetase was the generous gift of Dr. Edith Miles. 3R and 3S[3-3H]serines were kindly provided by Dr. Heinz Floss.

TABLE A

Step

Purification (n -fold)

Percentage yield

u/ml

mg/ml

u/mg

480

0.313

112

0.002

1

400 200 50

0.279 0.98

11 132

39

21

0.726

1.5

0.005 0.022 0.264 0.484

242

10

0.120 1.25

0.03 0.276

4.0 4.5

PWkQ

72 40 4.5

1.19

2.5

100

75 25

in

2 0.5

G-150" “Enzyme

TYPICALPURIFICATIONOFCYSTATHIONINESYNTHASE

Volume (ml)

Crude homogenate Acid supernatant fluid DEAE column Hydroxyapatite DEAE column 3 M urea

I

applied

to this

column

was pooled

from

several

preparations.

2000 2250

-

2

RAT

CYSTATHIONINE

to other protein. A 250-fold purified preparation gives a single band on acrylamide gel electrophoresis and at least 12 bands on SDS electrophoresis. In purifying the enzyme, separation from associated proteins is achieved by chromatography in 3.5 M urea. Substrate Speci&ity

and Inhibitors.

Cystathionine synthase also catalyzes the conversion of P-Cl-alanine to cystathionine. When serine was replaced by p-Cl-[14C]alanine on the TLC assay, a radioactive compound was detected which comigrated with cystathionine. Formation of cystathionine from ,&Cl-alanine was further confirmed by cellulose thinlayer chromatography with acetone/0.5% aqueous urea (6/4, v/v) as the mobile phase. At pH 7.6 Vfor cystathionine formation is approximately 50% of the V in the presence of serine. When [14C]threonine was substituted for serine, a radioactive product was formed which comigrates with cystathionine in the TLC system used TABLE

II

COMPETITIVE INHIBITORS WITH RESPECT TO SERINE IN THE CYSTATHIONINE SYNTHASE REACTION Km

Inhibitor or substrate DL-Serine DL-fl-Chloroalanine” L-Threonine DL-3,3,3-Trifluoroalanine DL-VinyIgIycine DL-Serine methyl ester Cyelopropylamine DL-Cyanogiytine Cystathionine

+Homocysteine

or K, (mM) -Homocysteine

K,

= 2

K,

K, K,

= 50 = 17

K,,, = 4

K, = 2 K, I 17

= 0.2

K, = 3 K, = 0.5

No inhibition No inhibition K, I 20 K, = 65

Note. Cystathionine synthase (sp act 3.0 u/mg) in 270 mM Tris.HCl, pH 8.60, was incubated at 37°C with 64 mM DL-homocysteine, varying [U-‘%]serine concentrations, and changing fixed inhibitor concentrations. The TLC assay was used in the absence of homocysteine the aH release assay was used. a 270 mM potassium phosphate buffer, pH 7.8.

SYNTHASE

701

MECHANISM

TABLE

III

COMPETITIVE INHIBITORS WITH RESPECT TO HOMOCYSTEINE IN THE CYSTATHIONINE SYNTHASE REACTION Inhibitor DL-Methionine L-Homoserine DL-2-Aminobutyric L-Cysteine S-Methyl-L-cysteine

acid

Kj (mM) 460 950 400 11 114

Note. L-[U-‘%]serine, 10 mM; other conditions as in Table I. DL-Homocysteine concentration was varied from 1 to 64 mM at changing fixed inhibitor concentration. Reaction was assayed with TLC assay.

for the assay and binds tightly to strong cation ion-exchange resins. It also comigrated with cystathionine on high-voltage paper electrophoresis at pH 1.9. Although the product is probably 3-methyl-cystathionine, it was not positively identified. V for product formation from threonine is 10% of cystathionine formation from serine. It has been reported (5) that other thiols can replace homocysteine in the cystathionine synthase reaction. We have extended this study to include amino acid analogs of homocysteine listed in Table III. When cysteine, [14C]serine, and enzyme were incubated, [14C]lanthionine could be identified by thin-layer chromatography, N-BuOH/acetic acid/Hz0 (12/3/5) and acetone/0.5% urea (614). At V, lanthionine is formed at 0.8% of the cystathionine formation rate using homocysteine as substrate. When 64 mM homoserine was substituted for homocysteine, a product was formed that cochromatographs with cystathionine in the TLC system used for the assay. This product, not positively identified, was formed at less than 4% of the cystathionine formation rate using homocysteine as substrate. Reaction Kinetics The rate of cystathionine formation was determined at pH 8.6 under the conditions given in Table II (TLC assay) as a function of serine and homocysteine concentration. Reciprocal plots yielded an intersection line pattern. For serine, V = 4.2 nm min-’

702

BORCSOK

AND

ABELES

Km = 2 mM, and for homocysteine, Km = 5.7 mM. These values are in close agreement with previously reported results (11). With homocysteine, above 64 mM, marked inhibition was observed.

Cystathionine synthase is generally stored in mercaptoethanol. Therefore, in order to eliminate the possibility that 3H exchange observed in the absence of homocysteine was due to residual mercaptoethanol, the enzyme was dialyzed overnight against Exchange of Substrate Ly-Hydrogen 100 vol of 0.1 M potassium phosphate A possible reason for the failure of p- buffer, pH 7.8, containing 10m3M EDTA. replacing enzymes to catalyze elimination The ratio of the 3H exchange rate in the reactions might be that labilization of the presence and absence of homocysteine was a-hydrogen does not occur in the absence identical to that of Table IV. of cosubstrates. This has been reported for An experiment was also performed to the P-replacing enzymes, cysteine lyase determine whether any 3H is conserved in and serine sulfhydrase (5). The release of the conversion [cu-3H]serine to cystathiotritium from [&H]serine as well as p- nine. Conversion of [a-3H]serine to cysta[a-3H]chloroalanine to solvent was therethionine was carried out under standard fore examined. The results are summaconditions at pH 8.6. No 3H was detected rized in Table IV. Cystathionine synthase in cystathionine, therefore less than 0.3% catalyzes 3H release from the (Y position of the 3H released is transferred to cysof these substrates in the presence and tathionine. absence of homocysteine. In all cases, 3H These experiments were carried out was released more rapidly in the presence with enzymes that were approximately of homocysteine. In the absence of ho- 50% pure. The question, therefore, arises mocysteine, no significant net reaction oc- whether the exchange reaction could be curs and therefore, exchange of the a-hydue to a contaminating enzyme. The rate drogen of serine with solvent protons of tritium release from [a-3H]serine relaoccurs. In the presence of homocysteine, tive to cystathionine formation was mea3H release to solvent is either equal to the sured during the course of the purificarate of cystathionine formation or more tion. The ratio was found to be 0.3 for rapid, depending upon reaction conditions. enzyme purified less than 30-fold, and 0.12 When 3H release is more rapid than cys- f 0.03 for all enzyme preparations that tathionine formation, exchange of the LY- were purified more than 30-fold. In addition, this ratio was constant for enzymes 3H of unreacted serine with solvent protons occurs. It has been shown that in the that had been inactivated to varying excase of serine sulfhydrase and cysteine tent with heat, hydroxylamine, or iodolyase, mercaptoethanol promotes the ex- acetamide. The Ki for inhibition by triwas 3 + 1 mM for the change of the a-hydrogen of serine (5). fluoroalanine TABLE

IV

RATES OF 3H RELEASE TO SOLVENT FROM [W~H]SERINE AND 8-Cl-[CX-3H]A~~~~~~ v, Cystathionine formation (rrmol/min)

V, 3H release without homocysteine (pmol/min)

Km, 3H release without homocysteine (mM)

DH

V, ‘H release with homocysteine (rrmol/min)

DL-Serine

8.60 7.80

0.53 + 0.03 0.75 f 0.06

0.50 + 0.03 0.28 + 0.01

0.06 f 0.01 0.25 + 0.01

0.2 2.0

DL-@-Chloroalanine

7.80

0.14 * 0.07

0.13 + 0.02

0.08 f 0.01

4.0

Substrate

Note. Reaction conditions: DL-[cY-3H]serine, 10 mb%,or DL-@-[&H]chloroalanine, 10 mM. Other conditions as in Table I. For reactions at pH 7.8,270 mM potassium phosphate buffer was substituted for Tris. HCl. ‘H release was measured as described under Methods (assay). Cystathionine formation was measured with TLC assay. V is corrected for enzyme concentration.

RAT

CYSTATHIONINE

exchange reaction and the catalytic reaction. These results establish that the 3H release from [cr-3H]serine and p-Cl-[cu3H]alanine, described above, is catalyzed by cystathionine synthase and is not due to a contaminating enzyme. Product Formation Homocysteine

in the Absence

of

The occurrence of tritium release from [a-3H]serine and /3-C1-[a-3H]alanine in the absence of homocysteine raised the possibility that pyruvate might be formed. When [a-14C]serine was added to cystathionine synthase in the absence of homocysteine, no detectable pyruvate formation occurred (Table V); i.e., the rate of pyruvate formation is less than 0.01% the rate of tritium release. It is possible that conversion of serine to pyruvate does not occur because -OH is a poor leaving group, which requires enzyme-catalyzed protonation to facilitate its departure and this protonation does not occur in the absence of the second substrate. Therefore, conversion of P-Cl-alanine to pyruvate and serine was also investigated. Protonation is not required to facilitate the elimination of Cl-. The data in Table V show that conversion of /3-Cl-alanine to pyruvate or serine proceeds at less than 5% the rate of tritium release. The possibility also must be considered that the small conversion of P-Cl-alanine to serine and/or pyruvate that was observed is due to a contaminating enzyme. These experiments establish that the rate of formation of serine and fl-Cl-alanine is significantly lower than the rate of carbanion formation as measured by tritium release, which represents a lower limit to the rate of carbanion formation. Stereochemistry of Displacement, OH Group of Serine

the fi-

We considered that the displacement reaction catalyzed by cystathionine synthase proceeds by direct displacement, as shown in Eq. [3]. According to this mechanism, an imine is formed through the interaction of the substrate with pyridoxal

SYNTHASE

703

MECHANISM TABLE

V

RATES OF PRODUCT FORMATION AND (U-HYDROGEN RELEASE FROM SERINE AND CHLOROALANINE

Productformation Substrate Serine Chloroalanine

=Hrelease (wnol/ min) 0.85+ 0.01 0.08* 0.01

Pyruvate (~molhnin)
Serine (fimol/min) 0.003

+ o.lm

Note.Cystathioninesynthaae,0.28unit (sp act 1.04u/mg) WBSincubatedwith 280rn~ potassiumphosphate,pH 7.8,and 12 IIIML-(U“Clfhloroalanineor 10 rn~ L-[U-“Cjaerine,in a total volumeof 250 rl for 407 min at 3l’C. To measuretritium release[w3H]serineor [w%]chloroalanine was substitutedfor the ‘C-labeled substrates. Reactionproceededfor 15 min. ‘H release,pyruvateand swine formation were determinedas describedunder Methods.Pyruvateand wine formation~88 also determinedin a parallel reactionin which enzymewas omitted.This valuewas subtractedfromthat obtainedin the enzymicreaction.

phosphate. The imine activates the /3 substituent toward direct displacement by the mercaptide anion. This mechanism does not involve an intermediate enamine. It is likely that a displacement mechanism proceeds with inversion of configuration at the P-carbon of serine. We, therefore, investigated the stereochemical course of the displacement of the P-OH group of serine by homocysteine. The series of reactions used to determine the stereochemistry of each reaction presented in Scheme 1 is well known (7) (with the exception of the cystathionine synthase reaction) including the inversion that accompanies the nonenzymatic conversion of S-methylcysteine to serine (Reaction IV, Scheme 1). 3R-[3-3H]Serine was converted to cystathionine with cystathionine synthase. Cystathionine was then converted to serine (Reactions II - IV, Scheme 1). After [14C]serine addition and determination of 3H/‘4C ratio, serine was converted to indolmycin (through Reactions V and VI, Scheme 1). The 3H/‘4C ratio of indolmycin was determined and the result presented in Table VI (Reaction A). Since 91% of the tritium was lost, the serine formed through reaction IV was 3S-[3-3H]serine. Hence, the displacement of the @hydroxyl group of serine when cystathionine is formed proceeds with retention of configuration. A similar experiment was performed with 3S-[3-3H]serine. The data in Table VI (Reaction B) show that the tritium is re-

704

BORCSOK

coon Ii

ABELES

coon

Cyrtothionine Synthore

H

AND

c

COOH

H

y-Cystothionase

H

I

L

H

OH

SCH2CH2FH

SH COOH

2S,3R-[3-3H]-Serine

COOH O,N-C,H,-S03CH3

L

2R.3R-[3-3H]-Cysteine

I) Ac20

COOH

2) BrCN

H

60%

I.! “H‘y”

Iu.

H

II

HCOOH

-

H

ISI SCH3

6H 2S,3S-

[3-3H]-Serine

COOH Tryptophon

Synthase P

PL H

H lndolmycm

2S,3f?-[3-3H]-Tryptophon

SCHEME

1. Conversion

of ZS, 3R-[3-3H]serine

tained in the [14C]indolmycin formed. These results are consistent with the results obtained with 3R-[3-3H]serine. A more direct method of determining the stereochemistry of the cystathionine synthase reaction involves the direct conversion of S-methylcysteine to tryptophan, as catalyzed by tryptophan synthase (21). However, the stereochemistry of the tryptophan synthetase reaction, using STABLE DETERMINATIONOFTHE

to indolmycin.

methylcysteine as substrate, has not been established. We performed this conversion using 3S-[3-3H]-S methylcysteine prepared from 3S-[3-3H]serine (Reactions I-III, Scheme 1). The results in Table VI (Reaction C) show that tritium is lost upon conversion to indolmycin. In view of the above results, it can be concluded that tryptophan formation from S-methylcysteine proceeds with retention of configuVI

STEREOCHEMISTRYOFTHEREACTIONCATALYZEDBYCYSTATHIONINESYNTHASE 3H/14C

Reaction A B C D

S-Methylcysteine 1.40 -

Serine 2.64

0.83 -

Tryptophan

Indolmycin

1.32 0.40 1.40 2.30

0.11 0.32 0.06 2.14

Percentage 3H retained 9 88 3 93

Note. Reaction A: The conversion of 3R-[3-3H]serine to cystathionine, then to indolmycin via Reactions II through VI, Scheme 1. Reaction B: The conversion of 3S-[3-3H]serine to cystathionine, then to indolmycin (Reaction II through VI, Scheme l).a Reaction C: The conversion of 3S-[3-%jmethylcysteine to tryptophan, then indolmycin. Reaction D: The conversion of 3R-[3-3H]serine to indolmycin via (Reactions V and VI, Scheme l).” a The reactions used are shown in Scheme 1. The steric configurations shown in Scheme 1 do not apply.

RAT

CYSTATHIONINE

ration as does tryptophan formation from serine. A control experiment was carried out to verify the stereochemical purity of 3R-[33H]serine used in these experiments. When 3R-[3-3H]serine was converted to indolmycin, 93% of the tritium was retained in indolmycin (Table VI, Reaction D). As both Reactions V and VI proceed with retention of configuration, it can be concluded that 3R-[3-3H]serine is stereochemically pure. Inactivators

of Cystathionine

Synthase

2-Amino-3-chloro-butenoic acid was tested as a potential inactivator. If this compound interacts with enzyme-bound pyridoxal, elimination of HCl could occur. This would lead to generation of a ketene at the active site. Precedent for this type of inactivation is provided by the inactivation of aminooxidase by 2-chloroalkylamines (22). Cystathionine synthase was not inactivated by this compound. It apparently binds poorly since its Ki = 250

mM.

Inactivation was achieved by 2-amino4-chlorobutyric acid. At pH 7.8 under standard conditions with 30 mM inactivator activity was lost with tl12 = 25 min. This inactivation was not studied in detail and the mechanism of this inactivation is not known. DISCUSSION

The results obtained establish these facts concerning the mechanism of action of cystathionine synthase: (i) The displacement of the P-OH group of serine proceeds with retention of configuration. The stereochemistry of the reaction catalyzed by cystathionine synthase is, therefore, the same as that of other pyridoxaldependent replacement reactions that have been studied (6-9). (ii) The reaction does not show ping-pong kinetics, but an intersecting line pattern. (iii) The enzyme catalyzes abstraction of the a-proton of serine as well as that of P-Cl-alanine in the absence of homocysteine as evidenced by exchange reactions. However, catalytic elim-

SYNTHASE

MECHANISM

705

ination of the ,&OH group or the ,&chloro group does not occur, or occurs at much slower rate than a-proton abstraction (carbanion formation). These results render several mechanisms unlikely. The mechanism shown in Eq. [2], in which the p substituent is released, and the elimination is completed prior to binding of homocysteine is not tenable, since such a mechansim would be associated with ping-pong kinetics, whereas an interesting line pattern was actually observed (23). This pattern is consistent with a mechanism in which no product release occurs until after the second substrate is bound. The direct displacement mechanism, as described in Eq. [3], also becomes unlikely since such a mechanism is expected to proceed with inversion of configuration. The displacement of the P-OH group of serine by homocysteine proceeds with retention of configuration. The stereochemical results are consistent with a front side displacement of the p substituent by homocysteine. However, since the /3 substituent is most probably present at the active site when the second substrate binds, a steric problem arises. This problem has been discussed in connection with tryptophan synthase (8). It was suggested that the ,&leaving group and the second substrate bind at different areas of the active site and that a conformational change occurs which causes the P-carbon of serine to shift from one area to the other. An additional mechanism would also be consistent with the stereochemical results (8). A covalent adduct is formed between the enzyme and the P-carbon of serine. The enzyme is then displaced by homocysteine. This double replacement reaction would also lead to retention of configuration at the P-carbon. We consider this mechanism unlikely since no evidence for this type of mechanism has even been obtained for pyridoxal phosphate-dependent enzymes. The results show that cystathionine synthase, in the absence of homocysteine catalyzes the exchange of the a-hydrogen of serine and P-chloroalanine. However, little or no /3 elimination (pyruvate formation) occurs. With serine no pyruvate

'706

BORCSOK

was detected and with P-chloroalanine the rate of pyruvate formation is less than 1.5% the rate of exchange of tritium of [a3H]chloroalanine. It is possible that elimination of the /3 substituent occurs but the carbon skeleton is retained on the enzyme. If that were the case, then /3-chloroalanine would be converted to serine. However, the rate of serine formation from P-chloroalanine is less than 5% the rate of exchange of the substrate a-tritium. Therefore, in the absence of homocysteine, cystathionine synthase catalyzes exchange of the o-hydrogen but not the elimination of the p substituent. Thus, although formation of an a-carbanion takes place, the ,L?substituent is not eliminated. Elimination occurs only in the presence of homocysteine. Homocysteine does not greatly accelerate carbanion formation, as indicated by the rate of cY-tritium exchange. Why then is the elimination reaction not completed in the absence of homocysteine? We have considered several mechanisms by which this can be achieved: (i) Elimination occurs concomitant with carbanion formation but the group which is eliminated is not released from the enzyme until homocysteine is bound. This mechanism requires that the enzyme binds OH- as well as Cl- at the active site, and that the elimination of HCl from P-Cl-alanine is reversible. Both of these requirements appear to us unreasonable, but cannot be ruled out. Therefore, this mechanism cannot be dismissed. (ii) In the absence of the second substrate, serine or ,&Cl-alanine is bound at the active site so that the leaving group is orthogonal to the CY-C-H bond. When the second substrate is bound a conformational change occurs which allows the leaving group to assume a syn or anti conformation. For nonconcerted elimination reactions, syn or anti conformations are required, whereas elimination from orthogonal conformation is highly unfavorable (24, 25). (iii) In the absence of homocysteine, the concentration of the serine-derived carbanion and hence the rate of elimination, could be reduced by providing another reaction pathway for the carbanion. This could involve proton-

AND

ABELES

ation of the C-5’ carbon of pyridoxal

(Eq.

[41):

e

“L-C4o g L \oI

According to this mechanism, a partial transamination reaction occurs in the absence of homocysteine, which prevents the elimination. There is some precedent for the proposal that an enzyme that is not a transaminase catalyzes a transamination reaction. Under certain conditions, tryptophan synthase catalyzes a transamination reaction (26) as well as aspartate @decarboxylase (27). However, no evidence has been obtained for a transamination reaction catalyzed by cystathionine synthase. With the available data it is not possible to distinguish among the three mechanisms outlined above. We tend to favor the second mechanism. Tryptophan synthase, an enzyme which catalyzes P-replacement reaction, shows properties similar to those which we have described for cystathionine synthase. The enzyme ((Y&J does not catalyze the conversion of serine to pyruvate or tryptophan to indole and pyruvate (28). This failure to catalyze elimination reactions is not due to inability to catalyze proton abstractions, since tryptophan synthase catalyzes the exchange of the a-hydrogen of tryptophan at approximately 20% of the normal synthetic reaction (8). The P subunit alone catalyzes the conversion of serine to pyruvate and chloroalanine to pyruvate. Thus it appears that the combination of (Y and /3 subunits brings about a conformational change, which prevents completion of the elimination reaction. It is obviously advantageous for enzymes whose primary role in metabolism is the catalysis of replacement reactions not to catalyze elimination reactions. This investigation of the mechanism of action of cystathionine synthase was motivated by our desire to develop a suicide inactivator for this enzyme and, if possible, to explain the failure of /3,&@-trifluoroalanine to inactivate cystathionine syn-

RAT

CYSTATHIONINE

thase. We have insufficient experimental information to answer this question. However, our data show that, in the absence of homocysteine, cystathionine synthase catalyzes elimination of the substrate ,6 substituent at a slow rate or possibly not at all. If the latter is the case, then p,/3,P-trifluoroalanine would not inactivate. REFERENCES 1. KIMURA, H., AND NAKAGAWA, H. (1971) J. B&hem. 69,711-723. 2. FINKELSTEIN, J. D. (1975) in Metabolic Pathways (Greenberg, D. M., ed.), Vol. VII, pp. 547-597, Academic Press, New York. 3. SILVERMAN, R. B., AND ABELES, R. H. (1976) Biochemistry 15,4718-4723. 4. BRAUNSTEIN, A. E. (1960) in The Enzymes (Boyer, P. D., ed.), Vol. 2, pp. 113-184, Academic Press, New York. 5. BRAUNSTEIN, A. E., AND GORYACHENKOVA, E. V. (1976) Biochimie 58, 5-17. 6. VEDERAS, J. C., SCHLEICHER, E., TSAI, M. D., AND FLOSS, H. G. (1978) J. Biol. Chem. 253, 53505354. 7. FLOSS, H. G., SCHLEICHER, E., AND Porrs, R. (1976) J. Biol. Chem. 251, 5478-5482. 8. TSAI, M. D., SCHLEICHER, E., AND POTPS, R. (1978) J. Biol Chem 253, 5344-5349. 9. CHEUNG, Y.-F., AND WALSH, C. T. (1976) J. Amer. Chem. Sot. 98, 3397-3398. 10. BERNARDI, G. (1971) in Methods in Enzymology (Jacoby, W. B., ed.), Vol. 17, pp. 325-339, Academic Press, New York. 11. SUDA, M., NAKAGAWA, H., AND KIMURA, H. (1973) in Methods in Enzymology (Tabor, M., and

SYNTHASE

MECHANISM

707

Tabor, C. W., eds.), Vol. 17, pp. 454-458, Academic Press, New York. 12. HERSHFIELD, M. S. (1979) J. Biol. Chem 254,22nr

‘0.

13. CZOK, R., AND LAMPRECHT, W. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd ed., Vol. 3, p. 1447, Academic Press, New York. 14. MILES, E. W., AND MCPHIE, P. (1974) J. BioL Chem. 249,2852-2857. 15. STEIN, W. H., AND MOORE, S. (1948) J. Biol. Chem 176, 367-388. 16. GREENSTEIN, J. P., AND WINITZ, M. (1961) in Chemistry of the Amino Acids, Wiley, New York. 17. FISHER, E., AND RASKE, K. (1907) Bw Dtsch Chem. Ges. 40, 3717-3724. 18. VALLE, G. J. V. (1965) Chem. A&r. 62, 4121. 19. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chm. 244,4406-4412. 20. HORNEMANN, U., HURLEY, L. H., SPEEDIE, M. K., AND FLOSS, H. G. (1971) J. Amer. Chem. Sot. 93, 3028-3035. 21. KUMAGAI, H., AND MILES, E. W. (1971) B&hem Biophys. Res. Commun 44,1271-1278. 22. KASHIWAMATA, S., AND GREENBERG, D. M. (1969) Fed. Proc. 28,668. 23. CLELAND, W. W. (1963) B&him. Biophys. Acta 67, 104-137. 24. SAUNDERS, W. H., AND COCKERILL, A. F. (1973) in Mechanisms of Elimination Reactions, Wiley, New York. 25. MULZER, J., AND KERKMANN, T. (1980) J. Amer. Chem Sot. 102, 3620-3622. 26. MILES, E. W., HATANAKA, M., AND CRAWFORD, I. P. (1968) Biochemistry 7, 2742-2753. 27. NOVOGRODSKY, A., NISHIMURA, J. S., AND MEISTER, A. (1963) J. Biol. Chem 238, PC 1903-1905. 28. KUMAGI, H., AND MILES, E. W. (1971) B&hem. Biophys. Res. Cwmmun 44, 1271-1278.