Yeast Cystathionine β-Synthase Is a Pyridoxal Phosphate Enzyme but, Unlike the Human Enzyme, Is Not a Heme Protein
2000; Elsevier BV; Volume: 275; Issue: 16 Linguagem: Inglês
10.1074/jbc.c000056200
ISSN1083-351X
AutoresKwang-Hwan Jhee, Peter McPhie, Edith Wilson Miles,
Tópico(s)Metabolism and Genetic Disorders
ResumoOur studies of cystathionine β-synthase fromSaccharomyces cerevisiae (yeast) are aimed at (1.Mudd S.H. Levy H.L. Skovby F. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 6th Ed. McGraw-Hill, New York1995: 1272-1327Google Scholar) clarifying the cofactor dependence and catalytic mechanism and (2.Chen P. Poddar R. Tipa E.V. Dibello P.M. Moravec C.D. Robinson K. Green R. Kruger W.D. Garrow T.A. Jacobsen D.W. Adv. Enzyme Regul. 1999; 39: 93-109Crossref PubMed Scopus (143) Google Scholar) obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine β-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine β-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with l-serine in the absence of l-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, l-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine β-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymesO-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase. Our studies of cystathionine β-synthase fromSaccharomyces cerevisiae (yeast) are aimed at (1.Mudd S.H. Levy H.L. Skovby F. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 6th Ed. McGraw-Hill, New York1995: 1272-1327Google Scholar) clarifying the cofactor dependence and catalytic mechanism and (2.Chen P. Poddar R. Tipa E.V. Dibello P.M. Moravec C.D. Robinson K. Green R. Kruger W.D. Garrow T.A. Jacobsen D.W. Adv. Enzyme Regul. 1999; 39: 93-109Crossref PubMed Scopus (143) Google Scholar) obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine β-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine β-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with l-serine in the absence of l-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, l-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine β-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymesO-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase. pyridoxal phosphate cystathionine β-synthase isopropyl thioglucoside phenylmethylsulfonyl fluoride N-α-p-tosyl-l-lysine chloromethyl ketone N-tosyl-l-phenylalanine chloromethyl ketone polymerase chain reaction kilobase pair(s) Elevated plasma homocysteine is an important risk factor in coronary heart disease and other human diseases (1.Mudd S.H. Levy H.L. Skovby F. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 6th Ed. McGraw-Hill, New York1995: 1272-1327Google Scholar, 2.Chen P. Poddar R. Tipa E.V. Dibello P.M. Moravec C.D. Robinson K. Green R. Kruger W.D. Garrow T.A. Jacobsen D.W. Adv. Enzyme Regul. 1999; 39: 93-109Crossref PubMed Scopus (143) Google Scholar, 3.Taylor Jr., L.M. Moneta G.L. Sexton G.J. Schuff R.A. Porter J.M. J. Vasc. Surg. 1999; 29: 8-21Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). One of the two major routes for detoxication of homocysteine is the pyridoxal phosphate (PLP)1-dependent β-replacement reaction with l-serine catalyzed by cystathionine β-synthase (CBS; EC 4.2.1.22). Lhomocysteine+Lserine→Lcystathionine+H2OEquation 1 The deduced sequences of human (4,5), rat (6.Swaroop M. Bradley K. Ohura T. Tahara T. Roper M.D. Rosenberg L.E. Kraus J.P. J. Biol. Chem. 1992; 267: 11455-11461Abstract Full Text PDF PubMed Google Scholar), andSaccharomyces cerevisiae (yeast) (7,8) CBS are similar. The finding that human CBS complements the cysteine auxotrophy of a yeast strain lacking CBS (5.Kruger W.D. Cox D.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6614-6618Crossref PubMed Scopus (69) Google Scholar) demonstrates the functional conservation of the human and yeast genes. The remarkable observation that the sequence of rat CBS (6.Swaroop M. Bradley K. Ohura T. Tahara T. Roper M.D. Rosenberg L.E. Kraus J.P. J. Biol. Chem. 1992; 267: 11455-11461Abstract Full Text PDF PubMed Google Scholar) is identical to the sequence of rat hemoprotein H-450 (9.Ishihara S. Morohashi K. Sadano H. Kawabata S. Gotoh O. Omura T. J. Biochem. (Tokyo). 1990; 108: 899-902Crossref PubMed Scopus (19) Google Scholar) led to the discovery that rat and human CBS contain both PLP and heme (10.Kery V. Bukovska G. Kraus J.P. J. Biol. Chem. 1994; 269: 25283-25288Abstract Full Text PDF PubMed Google Scholar). Heme may play a role in redox regulation of the human enzyme and in binding homocysteine (11,12). Although yeast CBS has been purified to homogeneity (13.Ono B. Kijima K. Inoue T. Miyoshi S. Matsuda A. Shinoda S. Yeast. 1994; 10: 333-339Crossref PubMed Scopus (29) Google Scholar), the absorption spectrum and cofactor content have not been reported. 2After this work was completed and reported in abstract and poster form at the 10th International Symposium of Vitamin B6 and Carbonyl Catalysis and 4th Meeting on PQQ and Quinoproteins, Santa Fe, New Mexico, October 31–November 5, 1999, we learned that another group had purified CBS from S. cerevisiae and found that the enzyme is not dependent on heme (K. N. Maclean, M. Janosik, J. Oliveriusova, V. Kery, and J. P. Kraus, submitted for publication). 2After this work was completed and reported in abstract and poster form at the 10th International Symposium of Vitamin B6 and Carbonyl Catalysis and 4th Meeting on PQQ and Quinoproteins, Santa Fe, New Mexico, October 31–November 5, 1999, we learned that another group had purified CBS from S. cerevisiae and found that the enzyme is not dependent on heme (K. N. Maclean, M. Janosik, J. Oliveriusova, V. Kery, and J. P. Kraus, submitted for publication). Here, we demonstrate that purified yeast CBS contains PLP but not heme. Because the absence of heme facilitates spectroscopic studies of the PLP and of enzyme-substrate intermediates, we are able to demonstrate directly that CBS converts l-serine to an aminoacrylate intermediate, as expected for a PLP enzyme that catalyzes a β-replacement reaction (14,15). l-Cystathionine andl-serine were from Fluka. δ-Aminolevulinic acid,l-homocysteine thiolactone, aprotinin, pepstatin A, leupeptin, benzamidine hydrochloride, TPCK, TLCK, and PMSF were from Sigma. Gigapite was from Seikagaku, Japan. l-Homocysteine was prepared from l-homocysteine thiolactone as described (16.Suda M. Nakagawa H. Kimura H. Tabor H. Tabor C.W. Methods in Enzymology. 17B. Academic Press, New York1973: 454-458Google Scholar, 17.Drummond J.T. Jarrett J. Gonzalez J.C. Huang S. Matthews R.G. Anal. Biochem. 1995; 228: 323-329Crossref PubMed Scopus (72) Google Scholar). l-[U-14C]Serine (160 mCi/mmol) was from NEN Life Science Products. To overexpress CBS from S. cerevisiae, we ligated the 1.5-kb PCR product containing the CBS gene from PSTR4-2 (cys4) 3PSTR4-2 (cys4) was a generous gift from Dr. Yolande Surdin- Kerjan. (18.Cherest H. Thomas D. Surdin-Kerjan Y. J. Bacteriol. 1993; 175: 5366-5374Crossref PubMed Google Scholar) and the restricted pTRC 99A vector (Amersham Pharmacia Biotech) to yield the expression vector pSEC (Fig. 1). The PCR product of CBS was designed using an upstream PCR primer (5′-pATGACTAAATCTGAGCAGCAGCAAGCC-3′), which starts the 5′-phosphorylated ATG, and a downstream PCR primer (5′-GTTTGCTTTTATCTGCAGCGTGGG-3′), which extends from 8 to 31 bases after the termination codon for the CBS open reading frame. The boldface bases are changes that introduce aPstI restriction site. PCR reactions were carried out in 50 μl with 2-min denaturation at 94 °C followed by 35 cycles of 1-min denaturation at 94 °C, 30-s annealing at 50 °C, and 1-min extension at 72 °C using Pfu DNA polymerase and the reaction conditions described in the Stratagene catalog. The ethanol-precipitated PCR products were solubilized with TE buffer, pH 8.0 (10 mm Tris-HCl containing 1 mm EDTA), restricted by PstI (Amersham Pharmacia Biotech), and isolated by 0.7% agarose (Life Technologies, Inc., Ultra Pure) gel electrophoresis. The putative 1.5 kb CBS gene was cut out of the gel and extracted using a Geneclean II kit (Bio 101, Inc.). The overexpression vector pTrc 99A was restricted by NcoI, mung-bean nuclease (19.Hasan N. Kim S.C. Podhajska A.J. Szybalski W. Gene. 1986; 50: 55-62Crossref PubMed Scopus (41) Google Scholar), and then by PstI. After each restriction step, the 4.2-kb product was purified by agarose gel electrophoresis and extraction as described above. The restricted pTrc 99A vector was ligated with the amplified CBS PCR product using T4 DNA ligase and transformed into host E. coli DH5α (Life Technologies, Inc.). The seven-base distance between the AGGA site (E. coli ribosome binding site) of pTrc99A and the CBS start codon gives a high yield of overexpression. Recombinant pSEC was isolated using a QIA plasmid kit (Qiagen), and the total DNA sequence of the 1.5-kb insert was confirmed by DNA sequence analysis (Biopolymer Core Facility, University of Maryland at Baltimore). Finally, pSEC was transformed into E. coli XL1-blue (Stratagene) for overexpression. Protein concentrations were determined by the Coomassie Blue protein assay reagent (Pierce) using bovine serum albumin as a standard or from the specific absorbance of purified CBS at 280 nm (A 280 0.1% = 0.94). 4K.-H. Jhee, P. McPhie, and E. W. Miles, manuscript in preparation. CBS activity was determined by a modification of a standard method (20.Kraus J. Packman S. Fowler B. Rosenberg L.E. J. Biol. Chem. 1978; 253: 6523-6528Abstract Full Text PDF PubMed Google Scholar). The reaction mixture, which contained 200 mm Tris-HCl, pH 8.6, 20 μm PLP, 0.25 mg/ml bovine serum albumin, 5 mml-[U-14C]serine (800 cpm/nmol), and CBS (0.02–0.1 μg) in 18 μl, was preincubated for 5 min at 37 °C. The reaction was initiated by adding 2 μl of 50 mmhomocysteine to 5 mm and was terminated after 10–15 min by adding 5 μl of 50% trichloroacetic acid. After the mixture was centrifuged for 3 min, 5 μl of the supernatant was applied to a cellulose thin layer chromatography plate (Kodak). The product,l-[14C]cystathionine, was separated froml-[14C]serine by ascending thin layer chromatography in 2-propanol/formic acid/H20 (80/6/20 v/v). Radioactivity of the product was determined by PhosphorImager (Molecular Dynamics). One unit of activity is defined as the production of 1 μmol of l-cystathionine/h at 37 °C. A 1-liter culture ofE. coli XL1-blue transformed with pSEC was grown at 37 °C in Super Broth (BioWhittaker or KD Medical) containing tryptone (12 g/liter), yeast extract (24 g/liter), glycerol (6.3 g/liter), K2HPO4 (12.5 g/liter), KH2PO4 (3.8 g/liter), δ-aminolevulinic acid (50 mg/liter), ampicillin (100 mg/liter), and 20 ml of 50-fold concentrated Vogel and Bonner minimal medium (21.Vogel H.J. Bonner D.M. J. Biol. Chem. 1956; 218: 97-106Abstract Full Text PDF PubMed Google Scholar). A 10% inoculum was added to the medium, and growth proceeded for ∼3–4 h until the OD650 reached 2.5. IPTG was added to 0.1 mm, and growth was continued at 30 °C for 14–18 h. We found that adding IPTG to cells at high density (OD650 = 2.5) gave a higher yield of enzyme than adding IPTG to cells at lower density. Growth in triple-indented Tunair flasks with loose fitting plastic caps (Shelton Scientific Manufacturing, Inc.) (22.Tunac J.B. J. Ferment. Bioeng. 1989; 68: 157-159Crossref Scopus (22) Google Scholar) gave the highest yield of cells. Cells were harvested by centrifugation, washed with 0.85% NaCl containing 1 mm dithiothreitol, resuspended in Buffer BP (50 mm sodium/bicine, pH 7.8, containing 10 mmEDTA, 10 mm β-mercaptoethanol, 0.1 mm PLP, 1 mm PMSF, 0.1 mm TLCK, 0.1 mm TPCK, 1 mg/liter aprotinin, 2 mg/liter leupeptin, 2 mg/liter pepstatin, and 1 mm benzamidine-HCl), and disrupted by passaging twice through a French press at 8,000 p.s.i. The suspension was centrifuged at 12,000 × g for 30 min. The supernatant (crude extract) had a specific activity of 253 units/mg. Eight ml of a 2% solution of protamine sulfate in Buffer BP was added dropwise to the 50-ml crude extract with stirring at room temperature followed by additional stirring for 20 min. The suspension was centrifuged, and the precipitate was discarded. The supernatant solution was fractionated with ammonium sulfate at pH 7.5. The 30–60% ammonium sulfate fraction was dialyzed against three changes of Buffer BP at 4 °C for 6 h. The dialyzed enzyme solution (42 ml) was loaded onto a 2.5 × 20-cm column of DEAE-Sephacel, which was then washed with 300 ml of Buffer BP. The enzyme was eluted with a 1-liter linear gradient from 0 to 0.5 m NaCl in Buffer BP. Fractions were analyzed by SDS-polyacrylamide gel electrophoresis and by activity assay. The active fractions, which eluted between 0.18–0.25 m NaCl, were pooled, concentrated, and dialyzed against Buffer KP (10 mm potassium phosphate, pH 7.8, containing 10 mm EDTA, 10 mmβ-mercaptoethanol, 0.1 mm PLP, 1 mm PMSF, 0.1 mm TLCK, 0.1 mm TPCK, 1 ml/liter aprotinin, 2 mg/liter leupeptin, 2 mg/liter pepstatin, and 1 mmbenzamidine-HCl at pH 7.8). The dialyzed DEAE fractions had a specific activity of 438 units/mg (yield = 64%). The dialyzed enzyme solution (40 ml) was applied to a Gigapite column (3.1 × 27 cm) equilibrated with Buffer KP. Gigapite is a modified form of hydroxyapatite that has large particles and gives a high flow rate. The column was washed with 400 ml of Buffer KP followed by 300 ml of Buffer KP that contained 50 mm potassium phosphate. The enzyme was eluted with a 1.8-liter linear gradient ranging from 50 to 400 mm potassium phosphate in Buffer KP. The active fractions, which eluted at 150–200 mm potassium phosphate, were concentrated to 10–30 mg/ml, dialyzed against Buffer K (50 mm potassium phosphate, pH 7.5, containing 1 mmEDTA, 1 mm dithiothreitol, and 0.02 mm PLP), and stored at −85 °C. The Gigapite fractions had a specific activity of 470 units/mg (yield = 49%) and were >95% pure by the criterion of SDS-polyacrylamide gel electrophoresis. Approximately 600 mg of homogenous CBS was obtained from a 1-liter culture. All procedures were completed within 72 h to limit proteolysis. Absorption spectra of CBS were made using a Hewlett Packard 8452-diode array spectrophotometer thermostatted at 25 °C by a Peltier junction temperature-controlled cuvette holder. CD measurements (mean residue ellipticity in degree cm2/dmol) were made at 25 °C in a Jasco J-715 spectrophotometer interfaced with a personal computer (Japan Spectroscopic Co., Easton, MD). Our new expression vector, pSEC (Fig.1), gave a very high level of expression of the yeast CBS gene in E. coli (see "Experimental Procedures"). A 2-fold purification of yeast CBS by DEAE-Sephacel and Gigapite chromatography yielded homogeneous CBS in approximately 50% yield. Although yeast CBS has been purified previously (13.Ono B. Kijima K. Inoue T. Miyoshi S. Matsuda A. Shinoda S. Yeast. 1994; 10: 333-339Crossref PubMed Scopus (29) Google Scholar), the absorption spectrum was not reported.2 The absorption spectrum of our purified yeast CBS (Fig.2 A) exhibited major peaks at 280 and 412 nm in a ratio of 1: 0.16, typical of a PLP enzyme (15.Miles, E. W. (1986) in Pyridoxal Phosphate: Chemical, Biochemical and Medical Aspects (Dolphin, D., Poulson, D., and Avramovic, O., eds) Part B, Vol. 1, pp. 253–310, John Wiley and Sons, New YorkGoogle Scholar). Removal of the PLP resulted in an apoenzyme having no absorbance in the visible range.4 In contrast, the absorption spectra of rat and human CBS (10.Kery V. Bukovska G. Kraus J.P. J. Biol. Chem. 1994; 269: 25283-25288Abstract Full Text PDF PubMed Google Scholar, 11.Taoka S. West M. Banerjee R. Biochemistry. 1999; 38: 2738-2744Crossref PubMed Scopus (95) Google Scholar, 12.Kery V. Poneleit L. Meyer J.D. Manning M.C. Kraus J.P. Biochemistry. 1999; 38: 2716-2724Crossref PubMed Scopus (58) Google Scholar,23.Bukovska G. Kery V. Kraus J.P. Protein Expression Purif. 1994; 5: 442-448Crossref PubMed Scopus (70) Google Scholar, 24.Kery V. Poneleit L. Kraus J.P. Arch. Biochem. Biophys. 1998; 355: 222-232Crossref PubMed Scopus (136) Google Scholar, 25.Taoka S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 26.Taoka S. Widjaja L. Banerjee R. Biochemistry. 1999; 38: 13155-13161Crossref PubMed Scopus (81) Google Scholar) exhibit a visible absorption band at 428 nm, which is approximately equal in intensity to the 280 nm absorption band. The 428 nm band in rat and human CBS is attributed to the presence of both heme and PLP, which have overlapping visible absorption spectra in the 410–430 nm region. The absence of heme in yeast CBS is not due to expression in E. coli; human CBS expressed in E. coli does contain heme. We added δ-aminolevulinate to theE. coli growth medium because the presence of this precursor of heme has been shown to increase the heme content of human CBS (27.Kery V. Elleder D. Kraus J.P. Arch. Biochem. Biophys. 1995; 316: 24-29Crossref PubMed Scopus (42) Google Scholar). The presence of heme in rat and human CBS is surprising because no other PLP enzyme has been reported to contain heme. Several PLP enzymes that catalyze β-elimination and β-replacement reactions exhibit a sequence similarity to rat, human, and yeast CBS (28.Grishin N.V. Phillips M.A. Goldsmith E.J. Protein Sci. 1995; 4: 1291-1304Crossref PubMed Scopus (339) Google Scholar). Three of these related enzymes, O-acetylserine sulfhydrylase (29.Burkhard P. Rao G.S.J. Hohenester E. Schnackerz K.D. Cook P.F. Jansonius J.N. J. Mol. Biol. 1998; 283: 121-133Crossref PubMed Scopus (179) Google Scholar), the β-subunit of tryptophan synthase (30.Hyde C.C. Ahmed S.A. Padlan E.A. Miles E.W. Davies D.R. J. Biol. Chem. 1988; 263: 17857-17871Abstract Full Text PDF PubMed Google Scholar), and threonine deaminase (31.Gallagher D.T. Gilliland G.L. Xiao G. Zondlo J. Fisher K.E. Chinchilla D. Eisenstein E. Structure. 1998; 6: 465-475Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), have been analyzed by x-ray crystallography and shown to exhibit structural similarity. The spectroscopic properties of PLP provide a sensitive probe for detecting chemical intermediates in PLP-dependent β-replacement reactions (Scheme FS1) (15.Miles, E. W. (1986) in Pyridoxal Phosphate: Chemical, Biochemical and Medical Aspects (Dolphin, D., Poulson, D., and Avramovic, O., eds) Part B, Vol. 1, pp. 253–310, John Wiley and Sons, New YorkGoogle Scholar) and in other PLP-dependent reactions. The absence of heme in yeast CBS facilitates spectroscopic studies to detect intermediates in the reactions of CBS and to probe the reaction mechanism. The addition of l-serine to yeast CBS resulted in the disappearance of the 412 nm band attributed to the internal aldimine (E in Scheme FS1) and the appearance of a new spectroscopic species with a major band centered at 460 nm and a shoulder at 330 nm (Fig. 2 A). The 460 nm band is likely due to the aldimine of aminoacrylate (E-AA in Scheme FS1), which has been detected in the reaction of O-acetylserine sulfhydrylase withO-acetyl-l-serine (32.Cook P.F. Wedding R.T. J. Biol. Chem. 1976; 251: 2023-2029Abstract Full Text PDF PubMed Google Scholar, 33.Cook P.F. Hara S. Nalabolu S. Schnackerz K.D. Biochemistry. 1992; 31: 2298-2303Crossref PubMed Scopus (62) Google Scholar, 34.Woehl E.U. Tai C.H. Dunn M.F. Cook P.F. Biochemistry. 1996; 35: 4776-4783Crossref PubMed Scopus (58) Google Scholar) and ofd-serine dehydratase with d-serine (35.Schnackerz K.D. Ehrlich J.H. Giesemann W. Reed T.A. Biochemistry. 1979; 18: 3557-3563Crossref PubMed Scopus (64) Google Scholar). The 330 nm shoulder may be due to a different tautomer of E-AA, which is the predominant intermediate in the reaction of the closely related tryptophan synthase with l-serine (36.Drewe W.J. Dunn M.F. Biochemistry. 1985; 24: 3977-3987Crossref PubMed Scopus (95) Google Scholar). Our results (Fig.2 A) demonstrate the E-AA intermediate by direct absorption spectroscopy for the first time. Previous studies of the reaction of truncated or full-length human CBS with l-serine detected a putative aminoacrylate intermediate by difference absorption spectroscopy (12.Kery V. Poneleit L. Meyer J.D. Manning M.C. Kraus J.P. Biochemistry. 1999; 38: 2716-2724Crossref PubMed Scopus (58) Google Scholar) or by fluorescence spectroscopy (fluorescence emission at ∼400 nm with excitation at 330 nm) (11.Taoka S. West M. Banerjee R. Biochemistry. 1999; 38: 2738-2744Crossref PubMed Scopus (95) Google Scholar). Rapid scanning spectroscopy may be needed to detect the external aldimine intermediate (E-Ser in Scheme FS1) in the reaction of CBS withl-serine or intermediates in the reaction of CBS withl-serine and l-homocysteine. The addition ofl-homocysteine to CBS in the presence ofl-serine under the conditions shown in Fig. 2 Aresulted in a transient decrease in absorbance at 460 nm (data not shown), providing additional evidence that the 460 nm band is due to the E-AA intermediate. The reaction of the substrate analogl-alanine with yeast CBS yielded a band at 420 nm, which is a wavelength characteristic of the expected external aldimine, E-Ala (15.Miles, E. W. (1986) in Pyridoxal Phosphate: Chemical, Biochemical and Medical Aspects (Dolphin, D., Poulson, D., and Avramovic, O., eds) Part B, Vol. 1, pp. 253–310, John Wiley and Sons, New YorkGoogle Scholar). E-Ala is analogous to E-Ser in Scheme FS1. Yeast CBS exhibited a positive CD band centered in the visible absorption band of the cofactor at 412 nm (Fig. 1 B), as reported for O-acetylserine sulfhydrylase (37.Schnackerz K.D. Tai C.H. Simmons 3rd, J.W. Jacobson T.M. Rao G.S. Cook P.F. Biochemistry. 1995; 34: 12152-12160Crossref PubMed Scopus (65) Google Scholar) and tryptophan synthase (38.Miles E.W. Moriguchi M. J. Biol. Chem. 1977; 252: 6594-6599Abstract Full Text PDF PubMed Google Scholar, 39.Lane A.N. Kirschner K. Eur. J. Biochem. 1983; 129: 571-582Crossref PubMed Scopus (92) Google Scholar). The addition of l-serine gave a negative CD band centered at 460 nm and a strong positive band at 280 nm. Negative visible CD bands for E-AA have been reported forO-acetylserine sulfhydrylase (37.Schnackerz K.D. Tai C.H. Simmons 3rd, J.W. Jacobson T.M. Rao G.S. Cook P.F. Biochemistry. 1995; 34: 12152-12160Crossref PubMed Scopus (65) Google Scholar) and tryptophan synthase (39.Lane A.N. Kirschner K. Eur. J. Biochem. 1983; 129: 571-582Crossref PubMed Scopus (92) Google Scholar, 40.Kayastha A.M. Sawa Y. Nagata S. Miles E.W. J. Biol. Chem. 1991; 266: 7618-7625Abstract Full Text PDF PubMed Google Scholar). The tryptophan synthase E-AA intermediate also has a strong positive band at 280 nm (40.Kayastha A.M. Sawa Y. Nagata S. Miles E.W. J. Biol. Chem. 1991; 266: 7618-7625Abstract Full Text PDF PubMed Google Scholar). The addition of l-alanine gave a negative CD band centered at 430 nm. To probe the reversibility of the postulated CBS reaction in Scheme I, we measured the absorption spectra (Fig.3 A) and CD spectra (Fig.3 B) of yeast CBS in the presence ofl-cystathionine. The initial (15 s) absorption spectrum showed a peak at 430 nm and a prominent shoulder at 460 nm. The absorbance at 460 nm decreased with time (inset, Fig.3 A). The CD spectrum (∼6 min) exhibited a negative band at 460 nm and positive bands at 400 and 280 nm. Thus,l-cystathionine appears to undergo the reverse reaction to form E-AA and an aldimine, either the internal aldimine, E, or an external aldimine, E-Ser or E-Cyst (Scheme FS1). Our results provide the first direct, spectroscopic evidence that the reaction catalyzed by yeast CBS proceeds through the postulated intermediates in Scheme I and that the reaction is at least partially reversible. The absorption spectrum in Fig. 2 A showed thatl-serine was largely or completely converted to E-AA in the absence of l-homocysteine. This result demonstrates that the CBS reaction does not proceed by direct displacement of the OH ofl-serine, as proposed by Braunstein and Goryachenkova (41.Braunstein A.E. Goryachenkova E.V. Adv. Enzymol. 1984; 56: 1-89Crossref PubMed Google Scholar). Our results, therefore, resolve an old controversy over the mechanism of CBS (14.Borcsok E. Abeles R.H. Arch. Biochem. Biophys. 1982; 213: 695-707Crossref PubMed Scopus (53) Google Scholar, 15.Miles, E. W. (1986) in Pyridoxal Phosphate: Chemical, Biochemical and Medical Aspects (Dolphin, D., Poulson, D., and Avramovic, O., eds) Part B, Vol. 1, pp. 253–310, John Wiley and Sons, New YorkGoogle Scholar, 41.Braunstein A.E. Goryachenkova E.V. Adv. Enzymol. 1984; 56: 1-89Crossref PubMed Google Scholar) and are consistent with the stereochemical data showing that displacement of the OH of l-serine proceeds with retention of configuration (14.Borcsok E. Abeles R.H. Arch. Biochem. Biophys. 1982; 213: 695-707Crossref PubMed Scopus (53) Google Scholar). The direct displacement mechanism of Braunstein and Goryachenkova (41.Braunstein A.E. Goryachenkova E.V. Adv. Enzymol. 1984; 56: 1-89Crossref PubMed Google Scholar) was proposed to explain the inability of CBS to catalyze the conversion of l-serine to pyruvate and NH3 by a β-elimination reaction. Our data provide evidence that CBS, like the tryptophan synthase α2β2 complex and O-acetylserine sulfhydrylase, forms a stable, enzyme-bound E-AA intermediate that does not undergo hydrolysis or further reaction in the absence of an added nucleophile. In conclusion, we have demonstrated that yeast CBS is a heme-independent, PLP enzyme and have carried out initial spectroscopic studies that establish the overall catalytic mechanism. Work is in progress to investigate the reaction kinetics, domain composition, oligomeric structure, and substrate and nucleophile specificity of yeast CBS.4 The absence of heme in the functional yeast enzyme shows that heme is not essential for catalysis and suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase.
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