Journal of Experimental Botany, Vol. 51, No. 347, pp. 985-993,
June 2000
© 2000 Oxford University Press
Cysteine synthase (O-acetylserine (thiol) lyase) substrate specificities classify the mitochondrial isoform as a cyanoalanine synthase
IACR-Rothamsted, Biochemistry and Physiology Department, Harpenden, Hertfordshire AL5 2JQ, UK
Received 27 September 1999; Accepted 21 January 2000
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Abstract |
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A cyanoalanine synthase and two isoforms (A, cytosolic and B, chloroplastic) of cysteine synthase (O-acetylserine (thiol) lyase) were isolated from spinach. N-terminal amino acid sequence analysis of the cyanoalanine synthase gave 100% homology for the determined 12 residues with a published sequence for the mitochondrial cysteine synthase isoform. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions, although with different efficiencies. Michaelis–Menten kinetics were observed for all three enzymes when substrate saturation experiments were performed varying O-acetylserine, chloroalanine and cysteine. Negative co-operative kinetics were observed for cysteine synthases A and B when substrate saturation experiments were performed varying sulphide and cyanide, compared with the Michaelis–Menten kinetics observed for cyanoalanine synthase. The exception was negative co-operativity observed towards sulphide for cyanoalanine synthase with O-acetylserine as co-substrate. The optimum sulphide concentration was dependent on the alanyl co-substrate used. The amino acid sequence similarity places these three enzymes in the same gene family, and whilst the close kinetic similarities support this, they also indicate distinct roles for the isoforms.
Key words: Cysteine synthase, O-acetylserine (thiol) lyase, cyanoalanine synthase, compartmentation, enzyme kinetics.
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Introduction |
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Cysteine synthase (O-acetylserine (thiol) lyase) (EC 4.2.99.8) catalyses the formation of cysteine from O-acetylserine and bisulphide. In addition, the enzyme may be involved in the global regulation of S-assimilation in higher plants, as the substrates and products (OAS, sulphide and cysteine) of cysteine synthase have been implicated in the regulation of gene expression of the sulphate transporter (Smith et al., 1997
). At least three isoforms of cysteine synthase have been identified in higher plants by chromatographic separations and by isolation of cDNAs (Ikegami et al., 1993; Kuske et al., 1994, 1996; Rolland et al., 1992, 1993; Saito et al., 1992, 1993, 1994a, b; Warrilow and Hawkesford, 1998). The isoforms are located in the cytosolic, chloroplastic and mitochondrial compartments as determined by subcellular fractionation techniques followed by resolution by ion-exchange or hydrophobicity chromatography (Rolland et al., 1992; Kuske et al., 1996). The gene databases currently contain around 24 distinct entries for plant cysteine synthases, including three for spinach and ten for Arabidopsis. Phylogenetic analysis indicates that some of these fall into groups which coincide with subcellular locations (Nakamura et al., 1999). The need for these multiple isoforms and individual subcellular locations is not clear, although a requirement for cysteine synthesis in individual compartments to compensate for a lack of intracellular transport has been suggested (Lunn et al., 1990). Further detailed analysis of the isoforms is clearly required.
Kinetic studies on cysteine synthase (Bertagnoli and Wedding, 1977; Cook and Wedding, 1976; Ikegami et al., 1988a, b; Kuske et al., 1994; Rolland et al., 1996; Tai et al., 1993, 1995) have reported Michaelis–Menten kinetics (Tai et al., 1993, 1995; Betagnolli and Wedding, 1977) or allosteric (positive co-operativity) kinetics towards both substrates (Kuske et al., 1994; Rolland et al., 1996). The data presented in this paper further elaborates on the kinetic mechanisms.
Cyanoalanine synthase (EC 4.4.1.9) catalyses the formation of cyanoalanine from cysteine and cyanide with the liberation of free bisulphide. Cyanoalanine synthase is widespread amongst higher plants, with a proposed function of detoxifying HCN that arises from ethylene biosynthesis (Wurtele et al., 1985). Only one major form of cyanoalanine synthase has been described in leaves (Ikegami et al., 1988c, d; Hendrickson and Conn, 1969), however, two forms have been identified in germinating seeds (Hasegawa et al., 1995). Cyanoalanine synthase activity has been localized mainly to mitochondria (Wurtele et al., 1985; Hendrickson and Conn, 1969) and has an apparent relative molecular mass of 53 000 to 60 000 (Ikegami et al., 1988b, c; Hendrickson and Conn, 1969). Preliminary kinetic studies have shown that cyanoalanine synthase obeys Michaelis–Menten kinetics (Hendrickson and Conn, 1969).
Cysteine synthase and cyanoalanine synthase may be classified into the ß-substituted alanine synthase family of enzymes and show considerable similarity in the reactions they catalyse. In this paper it is shown that the major cyanoalanine synthase activity corresponds to the gene product of the cDNA attributed to mitochondrial cysteine synthase as there is strong sequence homology between the N-terminus of the cyanoalanine synthase isolated in this study and the published amino acid sequence for the mitochondrial isoform of cysteine synthase (Saito et al., 1994b). The kinetic characterization of two forms of cysteine synthase and the cyanoalanine synthase are described, showing that all of these enzymes are able to catalyse the same reactions under appropriate conditions, but that significant differences indicate indivdual roles in planta.
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Materials and methods |
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Chemicals
All chemicals were obtained from Sigma Chemical Company (Poole, UK) unless otherwise stated. Sodium phosphate, acetic acid, concentrated HCl, ammonium sulphate, EtOH, and KCN were supplied by BDH-Merck (Poole, UK). Sodium ascorbate and N,N-dimethyl-1,4-phenylene-diamine dihydrochloride were obtained from Aldrich Chemical Company (Poole, UK). DEAE-Sepharose (fast-flow), Octyl-Sepharose CL4B, Phenyl-Sepharose ‘High-Substitution’, and Superdex-200 were supplied by Pharmacia (Uppsala, Sweden).
Enzyme purification for kinetic studies
Cyanoalanine synthase and cysteine synthases A and B were purified from 5–6-week-old, greenhouse-grown leaves of Spinacea oleracea L. cv. Medina. The purification procedures adopted were modifications of those previously described (Warrilow and Hawkesford, 1998). The soluble protein fraction was isolated from leaves (stored at -75 °C) and was concentrated by ammonium sulphate precipitation (40–80% saturation). Cyanoalanine synthase was separated from cysteine synthase by chromatography on DEAE-Sepharose using a linear 0–0.25 M NaCl gradient in 50 mM sodium phosphate, pH 7. Cysteine synthase activity was separated into two isoforms (A and B) by chromatography on Octyl-Sepharose using a linear 1–0 M ammonium sulphate gradient in 50 mM sodium phosphate, pH 7. Column eluates were assayed for both cysteine synthesis and cyanoalanine synthesis activities as previously described (Warrilow and Hawkesford, 1998). Enzymically-active fractions separated by native PAGE (Warrilow and Hawkesford, 1998) were further analysed by SDS-PAGE (Laemmli, 1970) after electro-elution. Enzymically-active bands were detected using KCN-Pb reagent (108 mM TRIS base, 5.7 mM cysteine, 1.3 mM lead (II) acetate, 3.1 mM KCN, pH 9.5) at room temperature for 5–30 min. These bands were excised and electro-eluted for 5 h at 40 mA in 0.1% (w/v) SDS, 25 mM TRIS base, and 192 mM glycine. The proteins recovered were subjected to SDS-PAGE and protein bands were detected with Coomassie blue R-250 or by silver staining (Hawkesford and Belcher, 1991).
Purification of cyanoalanine synthase for N-terminal sequencing
Cyanoalanine synthase (~54 mg protein) recovered from DEAE-Sepharose was further purified by chromatography on a Phenyl-Sepharose ‘High-Substitution’ column (25x1.6 cm) using a linear 1–0 M ammonium sulphate gradient in 50 mM sodium phosphate, pH 7. The cyanoalanine synthase-containing fractions (~3.7 mg protein), which eluted as a single peak at 0 M ammonium sulphate, were concentrated using a 50 ml ultrafiltration cell and Microcon-10 tubes (Amicon Stonehouse, UK). This concentrated enzyme preparation was separated by PAGE using a (4–15% precast gel, Bio-Rad, Hemel Hempstead, UK) and the cyanoalanine synthase band was visualized by staining with KCN-Pb reagent (see above). The stained band was excised and electro-eluted (Bio-Rad 422 electro-eluter) and concentrated using Amicon Microcon-10 concentration tubes. SDS-PAGE was performed using a 4–15% gel, as described above, with approximately 49 µg of protein. The protein from the preparative gel was electrophoretically transferred onto PVDF Immobilon-P membrane (Millipore). Transferred protein on the membrane was visualized with Coomassie Blue R-250 (0.1% (w/v) in 50% MeOH), followed by destaining in 50% MeOH. The stained cyanoalanine synthase band (~7 µg protein) was excised for N-terminal amino acid sequencing at the John Innes Centre (Norwich, UK) Protein Sequencing Unit.
Cysteine synthesis reaction
OAS or chloroalanine+sodium sulphide=cysteine+acetate or chloride. The standard assay contained 5 mM OAS or chloroalanine, 3 mM sodium sulphide, 10 mM dithiothreitol, and 0.1 M sodium phosphate, pH 8, in a total volume of 0.2 ml. The reaction, initiated by the addition of OAS or chloroalanine, was incubated for 10 min at 26 °C, and terminated by mixing 0.15 ml of the reaction mixture with 0.35 ml of acidic ninhydrin reagent (1.3% ninhydrin (w/v) in 1 : 4 concentrated HCl : glacial CH3COOH) (Gaitonde, 1967). Colour development (absorbance at 550 nm) required heating at 100 °C for 10 min, followed by cooling and the addition of 0.7 ml of ethanol. One enzyme unit is defined as the conversion of 1 nmol of OAS or chloroalanine into cysteine per minute under the stated assay conditions. Quantities of enzyme used per assay were approximately 0.4 µg protein for cysteine synthases A and B, and 12 µg protein for cyanoalanine synthase when OAS was used as substrate. When chloroalanine was used as the substrate approximately 2–4 µg protein were used per assay for all three enzymes. Kinetic determinations were also made with chloroalanine at pH 9.8 using 2-amino 2-methyl 1-propanol buffer instead of phosphate. pH-profiles were determined for the three enzymes over the pH range 5.0–11.5 using phosphate buffer (0.1 M) for the pH 5–8.5 range and 2-amino 2-methyl 1-propanol (0.1 M) for the pH 8.5–11.5 range.
Cyanoalanine synthesis reaction
Cysteine+KCN=cyanoalanine+bisulphide. The standard assay (modified from Hasegawa et al., 1994), contained 1 mM cysteine free base, 0.75 mM KCN (cyanoalanine synthase) or 3 mM KCN (cysteine synthases A and B), and 160 mM 2-amino 2-methyl 1-propanol buffer, pH 9.8 in a 1 ml volume. The reaction mixture was incubated at 26 °C for 20 min prior to termination by the addition of 0.5 ml of acidic dye precursor solution (15 mM N,N-dimethyl-1,4-phenylene-diamine dihydrochloride, 3 mM iron (III) chloride, 4.2 M HCl). After standing for 20 min, the amount of Methylene-Blue dye formed was determined by measuring the absorbance at 745 nm. The amount of enzyme used per assay was 20–30 µg protein for cysteine synthases A and B and 2 µg protein for cyanoalanine synthase. One enzyme unit is defined as the conversion of 1 nmol of cysteine into cyanoalanine and bisulphide per minute under the stated assay conditions. pH-profiles were determined for the three enzymes over the pH range 7.0–11.5. 2-amino 2-methyl 1,3-propanediol (0.16 M) was used for the pH 7–9.5 range and 2-amino 2-methyl 1-propanol (0.16 M) was used for the pH 9.5–11.5 range.
In all assays the invariant substrate concentrations were chosen to be as close to the optimal values, which were determined separately, without risk of substrate inhibition effects. All experimental determinations were performed in triplicate except for the pH-profiles and optimal substrate concentration studies. For the determination of protein, the semi-micro Coomassie Blue dye-binding assay (Bio-Rad, Hemel Hempstead, UK) was used with BSA as standard.
Data analysis techniques
Kinetic parameters were determined by non-linear regression using the Michaelis–Menten equation to determine km values. The Hill equation was used to determine the k' and apparent Hill numbers (Segel, 1993). The single substrate inhibition equation (v=Vmax[S]/km+[S]2/ki+[S]) was used to fit data that showed signs of substrate inhibition, where ki is the inhibition constant. The analysis was performed using ProFit 5.01 (Cherwell Scientific, Oxford, UK).
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Results |
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Spinach cyanoalanine synthase was separated from the cysteine synthases by ion-exchange chromatography and two cysteine synthase isoforms A and B were resolved by hydrophobicity chromatography (Warrilow and Hawkesford, 1998
). Increases in specific activity of 23-, 24- and 32-fold were obtained for cyanoalanine synthase and cysteine synthases A and B, respectively, compared with the initial 40–80% ammonium sulphate fraction from spinach leaves. Chromatography of cyanoalanine synthase on Phenyl-Sepharose resulted in a further 3.5-fold increase in specific activity over the DEAE-Sepharose eluted fraction.
Native PAGE of the cyanoalanine synthase fraction gave only one enzymically active band which after excision and electro-elution was found to contain one major (Mr of 33 500) and three minor peptides (Mrs of 92 300, 56 600 and 37 600) as resolved by SDS-PAGE. Staining the preparative PVDF blot with Coomassie Blue R-250 gave relative staining intensities of approximately 70% for the 33 500 peptide, 20% for the 92 300 peptide and 5% each for the 56 600 and 37 600 peptides. Previous studies (Ikegami et al., 1988c) have shown that spinach cyanoalanine synthase has a native Mr of 60 000 and a subunit size of 30 000. Therefore, the major peptide of Mr 33 500 was concluded to be the cyanoalanine synthase peptide and the N-terminal amino acid sequence was determined and compared to the sequences for cytosolic (Saito et al., 1992), chloroplastic (Saito et al., 1993; Rolland et al., 1993) and mitochondrial (Saito et al., 1994b) isoforms of cysteine synthase (Fig. 1). The comparison suggests that the mitochondrial form of cysteine synthase described by Saito et al. (Saito et al., 1994b) was the same enzyme as the cyanoalanine synthase described here.
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Two enzyme reactions were studied, the cyanoalanine synthesis reaction, in which a ß-substituted alanine (usually cysteine) was converted into cyanoalanine in the presence of cyanide, and the cysteine synthesis reaction, in which a ß-substituted alanine (usually OAS) was converted into cysteine in the presence of bisulphide.
The pH-profiles using the cysteine synthesis reaction were similar for all three enzymes whether OAS or chloroalanine was used as substrate (data not shown). The optimum pH range was between 9 and 11.5 yielding a constant reaction rate which rapidly decreased as the pH was lowered below 8, down to near zero at pH 6.5. The pH-profiles for the cyanoalanine synthesis reaction were similar for cysteine synthases A and B giving optimum pH values of 11. Cyanoalanine synthase, however, had a pH optimum of 10 and the activity observed fell sharply at higher pH (to just 15% at pH 11.5). Below pH 10 the observed activity fell sharply to nearly zero for all three enzymes as the pH was lowered to pH 7.5.
The optimum OAS concentration for the cysteine synthesis reaction (Fig. 2A) was 8 mM for cysteine synthases A and B compared with 20 mM for cyanoalanine synthase. Inhibition occurred at higher OAS concentrations. The optimum chloroalanine concentrations (Fig. 2B) were 3 mM, 20 mM and 8 mM for cyanoalanine synthase and cysteine synthases A and B, respectively. Higher chloroalanine concentrations caused the greatest inhibition of cyanoalanine synthase activity and the least inhibition of cysteine synthase A. Substrate inhibition constants were determined and are shown in Table 1. All three enzymes had similar optimum sodium sulphide concentrations (Fig. 2C) of 5–6 mM when OAS was the co-substrate. At higher sulphide concentrations progressive inhibition occurred. Substrate inhibition constants could not be determined (by curve fitting) as the kinetics displayed deviated too far from the conventional Michaelis–Menten model. The optimum sodium sulphide concentrations were different when chloroalanine was used as the co-substrate (Fig. 2D). Cysteine synthases A and B showed an optimum sulphide concentration above 24 mM compared with 3–6 mM for cyanoalanine synthase. Higher sulphide concentrations resulted in some inhibition of cyanoalanine synthase.
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) and cysteine synthase B (X) using the single-substrate inhibition equation except for the sodium sulphide determinations. Rates shown are for cysteine synthases (v) or cyanoalanine synthase (v *) and are not directly comparable as protein used per assay is variable (see Materials and methods).
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Cysteine synthases A and B had similar optimum cysteine concentrations for the cyanoalanine synthesis reactions (Fig. 2E
) of 0.8–1.2 mM with inhibition occurring at higher cysteine concentrations. Cyanoalanine synthase had an optimum cysteine concentration of 1–2 mM with inhibition at higher cysteine concentrations that was less severe than for cysteine synthases A and B. Substrate inhibition constants for cysteine were determined (Table 1). A large difference in the optimum KCN concentrations was observed (Fig. 2F). Cysteine synthases A and B have optimum KCN concentrations above 120 mM and no inhibition was observed even at 800 mM concentrations (data not shown). In contrast, cyanoalanine synthase had an optimum KCN concentration of 0.6–1 mM with further increases in KCN concentration causing severe inhibition of the enzyme.
Variation of the substituted alanine substrate concentrations at fixed concentrations of nucleophilic substrate (cyanide and sulphide) gave kinetic patterns that closely matched the Michaelis–Menten model (Fig. 3). No allosterism was detected down to 0.2 mM for the cysteine synthesis reaction (Fig. 3A, B) and down to 0.02 mM for the cyanoalanine synthesis reaction (Fig. 3C). Hill numbers close to 1 were obtained (0.9–1.2). The km values for OAS, chloroalanine and cysteine using the three enzymes are shown in Table 1. Cyanoalanine synthase had a much lower affinity for OAS than cysteine synthase A or B. The km values for chloroalanine were similar for all three enzymes at pH 8 and pH 9.8 with cysteine synthase A having a km value only marginally higher than the other two enzymes. Cysteine synthase A had the highest affinity for cysteine closely followed by cysteine synthase B with cyanoalanine synthase having a 4-fold lower affinity for cysteine than cysteine synthase A.
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Allosteric kinetics were observed in the cysteine synthesis reaction when sulphide was the variable substrate (Fig. 4A
, B; Table 2), with the data fitting the Hill equation. All three enzymes had similar k' values (0.40–0.54) and Hill numbers (0.32–0.45) when OAS was the co-substrate. When chloroalanine was the co-substrate at pH 8 (Fig. 4C, D) all three enzymes gave similar k' values (0.47–0.87), but cyanoalanine synthase had a Hill number of 1.10 compared with 0.44 and 0.38 for cysteine synthases A and B. In the presence of chloroalanine, cyanoalanine synthase obeys Michaelis–Menten kinetics towards sulphide, but cysteine synthases A and B show allosteric negative co-operativity. The similar k' values for sulphide obtained whether OAS or chloroalanine was used at pH 8 suggests that the km for sulphide was independent of the substituted alanine substrate used as would be expected in a bi- bi- ping-pong reaction mechanism (Tai et al., 1993). Similar results were obtained by using chloroalanine as co-substrate at pH 9.8 although the k' values were 2–5-fold lower.
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The allosterism towards sulphide was reflected in the biphasic nature of Eadie–Hofstee plots (Fig. 4B
, D), with the first phase occurring at 0.018–0.6 mM sulphide and the second at 1.2–3 mM sulphide. The kinetic parameters derived for the two phases are shown in Table 2. All three enzymes had similar km1 values (0.02–0.03 mM) when OAS was used as the co-substrate. The km2 values for cyanoalanine synthase and cysteine synthase A were similar at 0.6 mM compared with 0.2 mM for cysteine synthase B. When chloroalanine was used as the co-substrate at pH 8, cysteine synthases A and B had similar km1 and km2 values whereas, cyanoalanine synthase had a km1 value that was 27-fold and 44-fold greater than those of cysteine synthases A and B, respectively. This difference is seen in the Eadie–Hofstee plot (Fig. 4D). The gradient of the two phases for cyanoalanine synthase are similar and in the direct linear plot (Fig. 4C), the velocity curve obtained closely resembled Michaelis–Menten kinetics. It is probable that the second phase observed at 1.2–3 mM sulphide for cyanoalanine synthase is due to substrate (sulphide) inhibition reducing the apparent slope of the Eadie–Hofstee plot. When chloroalanine was used as the co-substrate at pH 9.8, Eadie–Hofstee plots were monophasic for cyanoalanine synthase and biphasic for cysteine synthases A and B and were similar to Fig. 4D. The km1 values derived, however, were 2–5-fold lower than those determined at pH 8 and km2 values were also lower. Determinations at sulphide concentrations below 0.018 mM gave lower than expected v/[sulphide] values due to high levels of substrate utilization in the assay system leading to inaccurate determinations of the initial velocities in spite of correction using mean [S*] values (Segel, 1993).
Allosteric kinetics were also observed in the cyanoalanine synthesis reaction when KCN was the variable substrate with cysteine synthases A and B, but Michaelis–Menten kinetics were observed with cyanoalanine synthase (Fig. 4E; Table 2). Similar k' values for KCN were obtained with cysteine synthases A and B (9.9 and 6.9) compared with a much lower k' value with cyanoalanine synthase (0.032). The Hill numbers for cysteine synthases A and B were identical at 0.49 suggesting negative co-operativity compared with 1.03 for cyanoalanine synthase, suggesting that Michaelis–Menten kinetics were obeyed. These differences between cyanoalanine synthase and cysteine synthases A and B were evident in the Eadie–Hofstee plots (Fig. 4F), which gave a monophasic straight line for cyanoalanine synthase compared with biphasic curves for cysteine synthases A and B. The km for KCN using cyanoalanine synthase was 0.031 mM, which was more than 10-fold lower than the km1 values for cysteine synthases A and B. The km2 values for cysteine synthases A and B were at 240 and 620 mM.
The relative substrate turnovers (Table 3) confirm that cysteine synthases A and B catalyse the cysteine synthesis reaction most efficiently using OAS as the substrate, but can also utilize chloroalanine in this reaction at 3–6% the rate of OAS at pH 8. Cysteine synthases A and B are less efficient at catalysing the cyanoalanine synthesis reaction with relative substrate turnovers of just 0.5% and 0.4% (3 mM KCN) to 1.7% and 1.6% (0.8 M KCN). Cyanoalanine synthase gave the highest relative turnover using chloroalanine in the cysteine synthesis reaction at pH 8, followed by cysteine utilization in the cyanoalanine synthesis reaction (73%) at pH 9.8. Cyanoalanine synthase was less efficient at using OAS in the cysteine synthesis reaction with a relative turnover for OAS of just 2.4%. No measurable activity was observed when homocysteine was used as the substrate for the cyanoalanine synthesis reaction (data not shown).
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Previous subcellular fractionation studies (Warrilow and Hawkesford, 1998
) have shown that cyanoalanine synthase was predominantly located in mitochondria, cysteine synthase A in the cytosol and cysteine synthase B in chloroplasts, in agreement with previous reports (Rolland et al., 1992; Kuske et al., 1996; Hendrickson and Conn, 1969). The data presented show that spinach cyanoalanine synthase and cysteine synthases A and B can all catalyse the same reactions under the appropriate conditions, although with different efficiencies (Table 3).
The amino acid sequencing results for the putative cyanoalanine synthase (Fig. 1) show that this peptide has 100% homology to the published sequence for mitochondrial cysteine synthase (Saito et al., 1994b), at least over the first 12 residues. The data suggest that this enzyme is a cyanoalanine synthase rather than a cysteine synthase because the enzyme is far more efficient at utilizing cysteine as a substrate than OAS.
A function of cyanoalanine synthase is to detoxify HCN formed during ethylene biosynthesis (Wurtele et al., 1985). As the cyanoalanine synthase is capable of synthesizing cysteine, this could potentially fulfil the biosynthetic requirements of the mitochondrion and so there would be no biochemical need for a separate mitochondrial cysteine synthase. The relative balance of the cyanoalanine synthesis and cysteine synthesis reactions is likely to be controlled by changes in localized pH. The rate of cyanoalanine synthesis will be reduced progressively as the pH is lowered from 10 down to 8 whilst the rate of cysteine synthesis falls much more slowly. The availability of substrates, especially cyanide and bisulphide, could also influence the balance of the two synthetic routes at a given pH. For example, the presence of KCN inhibits the cysteine synthesis reaction catalysed by cyanoalanine synthase.
The kinetics displayed when KCN was the variable substrate showed the largest difference between the three enzymes, with cysteine synthases A and B displaying allosteric kinetics (n values of 0.49) compared with cyanoalanine synthase which obeyed Michaelis–Menten kinetics. Cyanoalanine synthase had a 10-fold higher affinity for KCN than cysteine synthases A and B, explaining why cyanoalanine synthase is much more efficient at performing the cyanoalanine synthesis reaction. Cysteine synthases A and B showed no sign of inhibition at cyanide concentrations up to 0.8 M, clearly showing the allosterism displayed towards KCN in the absence of any substrate inhibition. If KCN binds to the same site as sulphide in the cysteine synthesis reaction, then similarly steep Eadie–Hofstee plots would be expected using sulphide for cysteine synthases A and B. Biphasic Eadie–Hofstee plots were obtained, but the second steep phases were curtailed due to substrate inhibition by sulphide at concentrations above 3 mM, especially when using OAS as co-substrate. Cysteine synthase, therefore, must have at least three binding sites for sulphide, a high affinity catalytic site, a low affinity catalytic site and a potential inhibitory binding site.
The lack of allosterism displayed by cysteine synthases A and B towards the alanine substrates contradicts the findings of other authors (Bertagnolli and Wedding, 1977; Kuske et al., 1994; Rolland et al., 1996). Bertagnolli and Wedding showed that Phaseolus cysteine synthase displayed positive co-operativity towards sulphide but not OAS (Bertagnolli and Wedding, 1977). Kuske et al. demonstrated that Datura cysteine synthase showed positive co-operativity towards both OAS and sulphide (Kuske et al., 1994). Most recently Rolland et al. showed that spinach chloroplast cysteine synthase expressed in E. coli displayed strong positive co-operativity towards sulphide and OAS and derived km1 and km2 values of 3.5 and 0.3 mM for OAS and 3.3 and 0.13 mM for sulphide (Rolland et al., 1996). It is difficult to reconcile these differences. If sulphide concentrations are low in the cell, typically in the µM range (Rosichan et al., 1983), then the first km should ideally be very low. This would allow the efficient utilization of sulphide in cysteine synthesis. The second km should be relatively high to avoid wasteful conversion of carbon skeleton alanines into cysteine when sulphur supply is in excess. Therefore, a negative co-operative allosteric model for cysteine synthase towards sulphide makes sense on a nutritional level compared with a positive co-operative allosteric model which would lead to low sulphide utilization at low cytosolic concentrations of sulphide and to wasteful conversion of OAS to cysteine at high concentrations of cytosolic sulphide. The positive co-operativity model would be beneficial if the primary function of cysteine synthase was to remove toxic sulphide from the cell, but would be of little use when sulphur concentrations were low. Rolland et al. explained this paradox by suggesting that substrate channelling for sulphide could occur (Rolland et al., 1996). Channelling inorganic bisulphide to cysteine synthase would be difficult due to diffusion effects and some sort of carrier sulphide would be more plausible, but little evidence exists for the presence of such carrier sulphides in plants. A negative co-operative model, however, would not require substrate channelling as it would be efficient at utilizing inorganic sulphide at low concentrations. The possibility of cysteine synthases A and B, or even cyanoalanine synthase displaying allosterism at very low alanine substrate concentrations cannot be excluded. However, at such low substrate concentrations, the enzyme assays used here would not be sensitive enough to ensure accurate determinations of the initial rates. Other studies using higher plant (Murakoshi et al., 1985; Ng and Anderson, 1978; Ngo and Shargool, 1973) and Salmonella (Tai et al., 1993, 1995) cysteine synthases also concluded that Michaelis–Menten kinetics were observed towards both OAS and bisulphide. Typical km values for Salmonella cysteine synthase were 0.2–1.6 mM for OAS and 0.003–0.07 mM for sulphide compared with 2 µM to 3 mM for OAS and 0.02–0.43 mM for sulphide in higher plants. Kinetic studies on cyanoalanine synthase (Ikegami et al., 1988c, d; Hendrickson and Conn, 1969) have shown that Michaelis–Menten kinetics are obeyed towards cysteine (km 1.6–3.6 mM) and cyanide (km 0.50–0.73 mM).
Cysteine synthases are located in the cytosol, plastidic and mitochondrial fractions, however, it is not clear why there are cysteine synthase isoforms in these individual subcellular compartments, particularly as the complete sulphate reduction pathway is thought only to occur in the plastid. The data in this paper suggest that at least one isoform has substantially distinct biochemical properties. This isoform is primarily a mitochondrial cyanoalanine synthase that also possesses a cysteine synthesis capacity and as such would have a distinctive biosynthetic role compared with other cysteine synthases.
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Acknowledgments |
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This project was supported by grant from the Biochemistry of Metabolic Regulation in Plants Initiative of the Biotechnology and Biological Sciences Research Council of the United Kingdom. IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom. The authors thank Dr Mike Naldrett at the John Innes Centre for performing N-terminal amino acid sequencing on the cyanoalanine synthase peptide.
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Notes |
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1 To whom correspondence should be addressed. Fax: +44 1582 763010. E-mail: malcolm.hawkesford{at}bbsrc.ac.uk
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Abbreviations |
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OAS, O-acetyl-L-serine..
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References |
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