Journal of Experimental Botany, Vol. 53, No. 368, pp. 439–445, March 2002
Modulation of cyanoalanine synthase and O-acetylserine (thiol) lyases A and B activity by b-substituted alanyl and anion inhibitors Andrew G. S. Warrilow and Malcolm J. Hawkesford1 IACR-Rothamsted, Agriculture and Environment Division, Harpenden, Hertfordshire AL5 2JQ, UK Received 16 August 2001; Accepted 31 October 2001
Abstract
Introduction
The reaction mechanisms of three enzymes belonging to a single gene family are compared: a cyanoalanine synthase and two isoforms of O-acetylserine (thiol) lyase (O-ASTL) isolated from spinach (Spinacea oleracea L. cv. Medina). O-ASTL represents a major regulatory point in the S-assimilatory pathway, and the related cyanoalanine synthase, which is specific to the mitochondrial compartment, has evolved an independent function of cyanide detoxification. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions although with different efficiencies, and which may be explained by a single amino acid substitution in the substrate-binding pocket of the enzyme. Substituted alanine and nucleophillic inhibitors caused predominantly non-competitive inhibition, indicating binding to both E- and F-forms of the enzyme in a bi–bi ping-pong kinetic model. Michaelis–Menten kinetics were observed when the alanyl substrate was varied in the presence and absence of inhibitors. The use of alanyl inhibitors has shown that the alanyl half-cycle of both the cysteine synthesis and cyanoalanine synthesis reactions of cyanoalanine synthase and O-acetylserine (thiol) lyases are similar. This is in contrast to the results observed with nucleophillic inhibitors, which have shown that the mechanisms of anion binding and processing differ between cyanoalanine synthase and O-ASTLs.
The O-acetylserine (thiol) lyase (O-ASTL) (EC 4.2.99.8) gene family may be subdivided and categorized as organellar, cytosolic or cyanoalanine synthase (EC 4.4.1.9)like (Jost et al., 2000; Hatzfeld et al., 2000). At least two forms of O-ASTL are present in the leaves of spinach (Warrilow and Hawkesford, 1998), one located in the cytosol (form A), and another located in the chloroplast (form B). One major form of cyanoalanine synthase was found in spinach leaves (Warrilow and Hawkesford, 1998) and was located primarily in the mitochondria. Three independent reports have confirmed that the mitochondrial member of this family represents the cyanoalanine synthase activity (Maruyama et al., 2000; Warrilow and Hawkesford, 2000; Hatzfeld et al., 2000). The two O-ASTLs and the cyanoalanine synthase are able to catalyse both the cysteine synthesis and cyanoalanine synthesis reactions (Warrilow and Hawkesford, 2000), but with varying efficiency. O-ASTL catalyses the formation of cysteine from O-acetylserine (OAS) and bisulphide. OAS is provided by serine acetyl transferase. This reaction is the first point at which sulphur is incorporated into the carbon skeleton of amino acids in the sulphur assimilatory pathway of higher plants. The substrates and products (OAS, sulphide and cysteine) of O-ASTL have been implicated in the regulation of gene expression of components of the sulphur assimilatory pathway such as the sulphate transporters (Smith et al., 1997). As a point of convergence of the N and S assimilatory pathways, the serine acetyl transferaseuO-ASTL complex (cysteine synthase) represents an important site of control, with flux through this step controlled by multiple regulatory loops. Serine acetyl transferase is only active in the complexed form
Key words: Cyanoalanine synthase, cysteine synthase, O-acetylserine(thiol)lyase, sulphur metabolism.
1
To whom correspondence should be addressed. Fax: q44 (0)1582 763010. E-mail:
[email protected] Abbreviations: O-ASTL, O-acetylserine (thiol) lyase; OAS, O-acetyl-L-serine.
ß Society for Experimental Biology 2002
440
Warrilow and Hawkesford
whilst O-ASTL functions in the uncomplexed state (Bogdanova and Hell, 1997; Droux et al., 1998). With an excess supply of both S and OAS, the cysteine that accumulates feedback inhibits serine acetyl transferase activity (Saito et al., 1995). When S-supply is limiting and OAS accumulates the excess OAS disrupts the complex preventing further OAS formation. Run-away cysteine production in the presence of excess S-supply is prevented as sulphide is an inhibitor as well as a substrate for O-ASTL (Warrilow and Hawkesford, 2000). In addition, accumulation of sulphide may have a role in repression of gene expression such as seen for the sulphate transporter (Smith et al., 1997). Kinetic studies of O-ASTL (Bertagnolli 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 shown that O-ASTL displays traditional Michaelis–Menten kinetics in some studies (Tai et al., 1993, 1995; Bertagnolli and Wedding, 1977), whilst exhibiting allosteric (positive co-operativity) kinetics towards both substrates in other studies (Kuske et al., 1994; Rolland et al., 1996). These findings have shown that spinach O-ASTLs display Michaelis–Menten kinetics towards the alanyl substrate and negative co-operative allosterism towards the anionic substrate (bisulphide) (Warrilow and Hawkesford, 2000). An allosteric binding site on Salmonella O-ASTL has recently been discovered (Burkhard et al., 2000). Cyanoalanine synthase 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) and recycling of the reduced N (Hatzfeld et al., 2000). Only one major form of cyanoalanine synthase has been described in leaves (Ikegami et al., 1988c, d; Hendrickson and Conn, 1969) and has been shown to reside mainly in mitochondria (Wurtele et al., 1985; Hendrickson and Conn, 1969). Kinetic studies have shown that cyanoalanine synthase obeys Michaelis–Menten kinetics (Hendrickson and Conn, 1969). Both O-ASTL and cyanoalanine synthase belong to the b-substituted alanine synthase family of enzymes and show considerable similarity in the reactions they catalyse (Warrilow and Hawkesford, 2000). In the present work, a comparison of the physicochemical properties of representative members of these subgroups is described, together with an analysis of reactions of the enzymes with their substrates and the effect of the modulation of the enzyme activities by b-substituted alanyl and anion inhibitors. The data are discussed in relation to the conserved residues of the OAS binding pocket of the enzyme (Burkhard et al., 1998, 1999, 2000).
Materials and methods Chemicals
All chemicals were obtained from Aldrich, Sigma Chemical Company or BDH-Merck (Poole, UK) unless otherwise stated. Enzyme purification for kinetic studies Cyanoalanine synthase and O-ASTLs 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 as previously described (Warrilow and Hawkesford, 1998, 2000). Cysteine synthesis reaction OAS or chloroalanineqsodium sulphide £cysteineqacetate or chloride: The cysteine synthesis assays used have been described previously (Warrilow and Hawkesford, 2000), together with cysteine detection (Gaitonde, 1967). Cyanoalanine synthesis reaction
CysteineqKCN £cyanoalanineqbisulphide: The cyanoalanine synthesis assays used have been described previously (Warrilow and Hawkesford, 2000) and are modified versions of other methods (Hasegawa et al., 1994). Enzyme–substrate preincubation studies Solutions of cyanoalanine synthase (121 mg ml1), O-ASTL A (40 mg ml1) and O-ASTL B (39 mg ml1) were incubated with 30 mM sodium sulphide at room temperature and the residual activities were determined at regular intervals (2–21 min) using the standard cysteine synthesis reaction above. Solutions of the three enzymes were also incubated with 5 mM OAS at room temperature and the residual activities were determined at regular intervals (0.25 min and 2 min) as described above. The O-ASTL A and O-ASTL B solutions were incubated with OAS concentrations of 2.5–20 mM at room temperature and the residual activities were determined after 0.25 min and 2 min as described above. The pH of the enzyme-OAS preincubations was kept at c. 7.0 to prevent acidic precipitation of the enzymes. Inhibitor studies
Inhibitor studies were performed using both the cysteine and cyanoalanine synthesis reactions using OAS, chloroalanine and cysteine as substrates. Two types of Inhibitors were used. These were the substituted alanines, OAS, b-cyanoalanine, homocysteine, and b-chloroalanine along with the anions, sodium thiocyanate and KCN (cysteine synthesis only). These inhibitors were used at 3 mM in the assay system, except for chloroalanine which was also used at 0.3 mM in the cyanoalanine synthesis reaction for O-ASTLs A and B. All inhibitor study determinations were performed in triplicate. Protein determination
For the determination of protein, the semi-micro Coomassie Blue dye-binding assay (Bio-Rad, Hemel Hempstead, UK) was used with bovine serum albumin as standard. Data analysis techniques Kinetic parameters were determined by non-linear regression of direct linear plots of v against wS x, linear regression of Eadie– Scatchard plots (vuwS x against v), linear regression of Hanes– Woolfe plots (wS xuv against wS x) and where possible non-linear
Inhibitor studies on O-acetylserine (thiol) lyase and cyanoalanine synthase regression of direct linear plots using the Michaelis–Menten single substrate inhibition equation. The Km and Vmax values presented in Tables 1, 2 and 3 are mean values with the associated standard errors not exceeding "5%. The type of inhibition displayed was determined by analysis of Hanes–Woolfe plots of the data. The Ki determinations were made using the equations Vmax.app ¼ (Vmaxu1q(wI xuKi)) for non-competitive inhibition and Km.app ¼ (Kmu1q(wI xuKi)) for uncompetitive inhibition (Segel, 1993). The Hill equation was used as a test for allosterism. The analysis was performed using ProFit 5.01 for Apple Power Macintosh (QuantumSoft, Zurich, Switzerland).
441
Preincubation with 15 mM OAS resulted in the total deactivation of the enzyme. The length of preincubation with OAS did not affect the observed deactivation of O-ASTL A. O-ASTL B was only moderately deactivated by preincubation with OAS, but the inactivation observed was dependent on the preincubation time. Preincubation in 20 mM OAS caused at 48% deactivation of O-ASTL B after 0.25 min compared with a 79% deactivation after 2 min. The OAS concentrations at which 50% inactivation occurred were c. 9 mM for O-ASTL A and less than 2 mM for O-ASTL B.
Results The enzyme purification scheme used (Warrilow and Hawkesford, 1998, 2000) separated spinach cyanoalanine synthase from the two O-ASTL isoforms A and B. Two enzyme reactions were studied, the cyanoalanine synthesis reaction, in which a b-substituted alanine (usually cysteine) was converted into cyanoalanine in the presence of cyanide, and the cysteine synthesis reaction, in which a b-substituted alanine (usually OAS) was converted into cysteine in the presence of bisulphide. No measurable activity was observed when homocysteine was used as the substrate for the cyanoalanine synthesis reaction or when cyanoalanine was used as the substrate for the cysteine synthesis reaction (data not shown). Inactivation by preincubation with substrates
Preincubation of the three enzymes with 30 mM sodium sulphide for 21 min caused a 5%, 9% and 16% inactivation of O-ASTL B, O-ASTL A and cyanoalanine synthase, respectively (data not shown). Preincubation with 5 mM OAS (Fig. 1A) resulted in 8–12%, 30% and 54% inactivation of cyanoalanine synthase, O-ASTL A and of O-ASTL B, respectively, after 2 min. Inactivation by OAS was confirmed by preincubation experiments on O-ASTLs A and B using OAS concentrations from 0–20 mM, at two time intervals (Fig. 1B). O-ASTL A was severely inactivated by preincubation with OAS.
Fig. 1. Enzyme–substrate preincubation inhibition of enzyme activity. (A) Preincubation of O-ATSL A (circles), O-ASTL B (squares) and cyanoalanine synthase (triangles) with 5 mM OAS; (B) preincubation of O-ASTL A (circles) and O-ASTL B (squares) with variable concentrations of OAS for 0.25 min (filled symbols) or 2 min (open symbols).
Kinetics of inhibition by b-substituted alanines and anions
The kinetics displayed by all three enzymes in the cysteine and cyanoalanine synthesis reactions obeyed the Michaelis–Menten model. Non-linear regression using the Michaelis–Menten and Hill equations both gave good visual fits to the data sets with Hill numbers of 0.8–1.2, indicating that little if any co-operativity between enzyme subunits was occurring when OAS, chloroalanine or cysteine were the variable substrates. The kinetic parameters associated with the modulation of the cysteine synthesis and cyanoalanine synthesis reactions by the alanyl and anion inhibitors were determined (Tables 1, 2, 3). Two types of inhibition were observed, non-competitive and uncompetitive. In the cysteine synthesis reaction with OAS as the substrate, the alanyl inhibitors caused uncompetitive inhibition of cyanoalanine synthase and noncompetitive inhibition of O-ASTLs A and B, with the exception of chloroalanine. The reverse was true when chloroalanine was the substrate, with the alanyl inhibitors causing non-competitive inhibition of cyanoalanine synthase and uncompetitive inhibition of O-ASTLs A and B. In the cyanoalanine synthesis reaction, with cysteine as the substrate the alanyl inhibitors, cyanoalanine and homocysteine, caused non-competitive inhibition of cyanoalanine synthase and O-ASTL A, but uncompetitive inhibition of O-ASTL B. Cyanoalanine and homocysteine were strong inhibitors of the cysteine and cyanoalanine synthesis reactions causing 25–50% inhibition at 3 mM concentrations. Chloroalanine was an uncompetitive inhibitor and caused moderate inhibition of the cysteine synthesis reaction with OAS as the substrate, causing 30–40% inhibition. Chloroalanine was an acute uncompetitive inhibitor of the cyanoalanine synthesis reaction, causing 60–75% inhibition. OAS was a non-competitive inhibitor yielding variable effects in the cyanoalanine synthesis reaction, with no inhibition of cyanoalanine synthase, mild inhibition of O-ASTL A and strong inhibition of O-ASTL B being observed. OAS was also a strong inhibitor of cyanoalanine synthase when chloroalanine was the substrate in the cysteine synthesis reaction.
442
Warrilow and Hawkesford
KCN was a strong inhibitor of cyanoalanine synthase, with 50–60% inhibition being observed. KCN, however, did not inhibit the cysteine synthesis reactions catalysed by O-ASTLs A and B. The action of sodium thiocyanate was variable, strongly inhibiting (50–60%) the cyanoalanine synthesis reaction of O-ASTLs A and B, whilst only mildly inhibiting (15%) cyanoalanine synthase. Sodium thiocyanate inhibition of the cysteine synthesis reaction was also variable, inhibiting all three enzymes when OAS was the substrate, but only inhibiting cyanoalanine synthase when chloroalanine was the substrate.
Discussion The substituted alanines used as inhibitors can be regarded as both analogues of the substrates and the products. The nucleophillic anion inhibitors would be expected to behave as substrate analogues of sulphide and cyanide. Tai et al. have previously shown that the catalytic mechanism of Salmonella O-ASTL fits the bi–bi pingpong model (Tai et al., 1993). In this model, the enzyme exists in either an E-form (which is able to bind substrate 1, here the alanyl substrate), or following dissociation
Table 1. Inhibition studies on cysteine synthesis reaction with OAS as substrate Substrate saturation experiments were performed using the cysteine synthesis reaction in the presence of 3 mM inhibitor with OAS as the alanine substrate as described in Materials and methods. Kinetic parameters were derived and used to calculate Ki values. The types of inhibition found were non-competitive (NC), and uncompetitive (UC). Vmax has units of nmoles of product formed per minute. Inhibitor
Control Cyanoalanine Homocysteine Chloroalanine Potassium cyanide Sodium thiocyanate
Cyanoalanine synthase
O-ASTL A
O-ASTL B
Vmax
Km (mM)
Ki (mM)
Type
Vmax
Km (mM)
Ki (mM)
Type
Vmax
Km (mM)
Ki (mM)
Type
2.15 0.53 0.78 – 0.44 1.15
10.5 2.32 3.14 – 2.35 3.41
0.86 1.29 – 0.87 1.45
UC UC – UC UC
4.45 2.49 3.48 2.85 5.58 3.46
1.33 1.50 0.92 1.79 2.88 1.59
3.83 6.73 5.35 – 10.6
NC UC UC None NC
3.93 2.46 3.05 2.86 5.10 2.91
1.16 1.55 1.16 2.18 2.59 1.14
5.03 10.5 8.00 – 8.57
NC NC UC None NC
Table 2. Inhibition studies on cysteine synthesis reaction with chloroalanine as substrate Substrate saturation experiments were performed using the cysteine synthesis reaction in the presence of 3 mM inhibitor with chloroalanine as the alanine substrate as described in Materials and methods. Kinetic parameters were derived and used to calculate Ki values. The types of inhibition found were non-competitive (NC), and uncompetitive (UC). Vmax has units of nmoles of product formed per minute. Inhibitor
Control O-acetylserine Cyanoalanine Homocysteine Potassium cyanide Sodium thiocyanate
Cyanoalanine synthase
O-ASTL A
O-ASTL B
Vmax
Km (mM)
Ki (mM)
Type
Vmax
Km (mM)
Ki (mM)
Type
Vmax
Km (mM)
Ki (mM)
Type
2.57 1.61 1.71 1.97 1.19 1.93
0.57 0.54 0.74 0.46 1.36 0.49
5.00 5.93 9.90 2.59 9.04
NC NC NC Mixed NC
1.19 – 0.83 0.95 1.31 1.20
0.91 – 0.59 0.33 0.96 0.99
– 3.43 1.28 – –
– UC UC None None
1.64 – 1.06 1.35 1.64 1.60
0.94 – 0.56 0.55 0.61 0.88
– 4.34 4.27 – –
– UC UC None None
Table 3. Inhibition studies on cyanoalanine synthesis reaction with cysteine as substrate Substrate saturation experiments were performed using the cyanoalanine synthesis reaction in the presence of 3 mM inhibitor with cysteine as the alanine substrate as described in Materials and methods. Kinetic parameters were derived and used to calculate Ki values. The types of inhibition found were non-competitive (NC), and uncompetitive (UC). Vmax has units of nmoles of product formed per minute. Inhibitor
Control O-acetylserine Cyanoalanine Homocysteine Chloroalanine Sodium thiocyanate
Cyanoalanine synthase
O-ASTL A
O-ASTL B
Vmax
Km (mM)
Ki (mM)
Type
Vmax
Km (mM)
Ki (mM)
Type
Vmax
Km (mM)
Ki (mM)
Type
1.78 1.87 1.62 1.38 0.77 1.49
0.40 0.40 0.41 0.51 0.46 0.34
– 30.0 10.1 2.27 15.1
None NC NC UC NC
0.46 0.40 0.36 0.31 0.18 0.20
0.11 0.11 0.10 0.27 0.73 0.07
22.4 11.3 6.09 0.19 6.36
NC NC NC UC UC
0.56 0.31 0.40 0.34 0.21 0.24
0.25 0.45 0.17 0.14 0.72 0.11
3.61 6.07 3.97 0.18 2.27
NC UC UC UC UC
Inhibitor studies on O-acetylserine (thiol) lyase and cyanoalanine synthase
of product 1, as the F-form which binds the second substrate (here bisulphide or cyanide). The E-form of the enzyme is regenerated by the release of product 2. Addition of inhibitor whilst varying the alanyl substrate concentration in such a model would yield competitive, non-competitive or uncompetitive inhibition if the inhibitor bound to the E-form, E- and F-forms, or F-form of the enzyme, respectively. Implications of alanyl inhibitor studies
The addition of an alternative substituted alanine caused either non-competitive or uncompetitive inhibition. Tai et al. established that cysteine was a non-competitive inhibitor of Salmonella O-ASTL, binding to both E- and F-forms of the enzyme (Tai et al., 1993). The alanine inhibitors compete with the substrate for the OAS binding pocket on the E-form of the enzyme. The alanyl inhibitors may bind on the F-form of the enzyme directly to the aminoacrylate-PLP group through the carboxylate anion of the alanyl inhibitor, as demonstrated by the production of cysteinylcysteine (Tai et al., 1993), to an anion binding site (Tai et al., 1993), or to an allosteric binding site (Burkhard et al., 2000). Further investigations into the nature of the second alanyl binding site on these enzymes are required. The uncompetitive inhibition obtained with some alanyl inhibitors demonstrated that under certain circumstances the alanyl inhibitor molecules were effectively excluded from the E-form of the enzymes. The reason for this exclusion is not known. The OAS-binding pocket (a-carboxylate binding loop) proposed for the prokaryotic enzyme (Burkhard et al., 1998) includes four essential residues, ser69, gly70, asn71, and thr72. These residues are conserved for all of the plant O-ASTL proteins which have been sequenced to date, with the exception of the cyanoalanine synthase-like group which includes the spinach mitochondrial isoform discussed here, oas5 from Brassica juncea (Scha¨fer et al., 1998), the Arabidopsis Bsas3;1 isoform (Hatzfeld et al., 2000) and two rice sequences, rcs2 and rcs4 (Nakamura et al., 1999) which cluster only loosely with this subgroup (Bsas6 in Hatzfeld et al., 2000). Thr 72 is replaced by a methionine for the cyanoalanine synthase-like sequences and by a leucine in the rice sequences. In both substitutions the absence of a hydroxyl group in the sidechain would contribute to a weaker binding of OAS, and hence the modified substrate specificities of this group. The Met side-chain does not contain a hydroxyl group and is bulkier than the Thr side-chain which would make the alanyl substrate binding pocket more crowded than those of the O-ASTLs. This may account for the low affinity and poor catalytic rates observed with OAS as substrate, as the OAS molecule would be sterically hindered from binding efficiently to the alanyl substrate pocket of cyanoalanine synthase.
443
Hendrickson and Conn established that cyanoalanine synthase and O-ASTL from blue lupin could catalyse the formation of cyanoalanine from both cysteine and OAS in the presence of cyanide (Hendrickson and Conn, 1969). The presence of OAS in the cyanoalanine synthesis reaction would be expected to compete with the utilization of cysteine, an activity not detected, as there would be no bisulphide production (which is required for the cyanoalanine synthase assay system used here). However, the present results (Table 3) suggest that the inhibition caused by OAS to the cyanoalanine synthesis reaction mirrored the inactivation caused by preincubation of the three enzymes with OAS in the absence of sulphide (Fig. 1): O-ASTL B being strongly inhibited by OAS compared to the slight inhibition of O-ASTL A. Cyanoalanine synthase was not inhibited by OAS in the cyanoalanine synthesis reaction. OAS also moderately inhibited cyanoalanine synthase when chloroalanine was the substrate in the cysteine synthesis reaction, even though cyanoalanine synthase had a low affinity for OAS. Chloroalanine was a powerful inhibitor of the cyanoalanine synthesis reaction. The observed inhibition was mainly due to chloroalanine taking part in two enzymecatalysed reactions: combining with cyanide to form cyanoalanine, and combining with sulphide to produce cysteine which depletes the product being measured in the assay system. All three enzymes also have higher turnover rates with chloroalanine than cysteine (Warrilow and Hawkesford, 2000) leading to the observed inhibition. Cyanoalanine and homocysteine were effective inhibitors of all three enzymes in both the cyanoalanine and cysteine synthesis reactions through the formation of dead-end complexes at the E-form of the enzymes and also by interaction with the F-form of the enzymes. Implications of anion inhibitor studies
The anion inhibitors gave either uncompetitive or noncompetitive inhibition. Tai et al. suggested that sulphide bound to both E- and F-forms of O-ASTL and, therefore, non-competitive inhibition would be expected (Tai et al., 1993). Burkhard et al., however, found no direct evidence for a sulphide binding site on the F-form of the enzyme, with sulphide apparently diffusing directly into the aminoacrylate-PLP site (Burkhard et al., 1998, 1999, 2000). Sulphide also interacts with the free enzyme (E-form) and at an allosteric anion binding site on Salmonella O-ASTL (Burkhard et al., 2000). Tai et al. demonstrated that thiocyanate was an uncompetitive inhibitor in relation to OAS for Salmonella O-ASTL (Tai et al., 1993). The variable inhibition results obtained with sodium thiocyanate require further investigation to establish the mode of action of this inhibitor. KCN caused strong inhibition of cyanoalanine synthase in the cysteine synthesis reaction. Cyanoalanine synthase
444
Warrilow and Hawkesford
can readily catalyse the synthesis of cyanoalanine from OAS and cyanide (Hendrickson and Conn, 1969), therefore causing depletion of OAS in the assay mixture and so reducing the rate of cysteine formation from OAS. The observed inhibition was strong because cyanoalanine synthase has a high affinity for cyanide (Km 0.03 mM; Warrilow and Hawkesford, 2000), driving the reaction toward production of cyanoalanine from OAS rather than cysteine. KCN caused no inhibition of O-ASTLs A and B, probably because of the relatively low affinities that these two enzymes have for cyanide (Km 0.3–0.4 mM) compared with sulphide (Km 0.03 mM; Warrilow and Hawkesford, 2000), which precludes the efficient use of cyanide in the synthesis of cyanoalanine from OAS.
Conclusions These studies have shown that cyanoalanine synthase can catalyse both the cysteine synthesis and cyanoalanine synthesis reactions. In vivo the relative ratio of the two reactions is likely to be controlled by factors such as the availability of bisulphide and cyanide and the local pH. The ratio of the cysteine synthesis to cyanoalanine synthesis reaction will fall as the pH increases from 7 to 10 if cyanide is present, with cyanoalanine synthesis becoming more prominent as the pH rises (Warrilow and Hawkesford, 2000). Therefore, the biosynthetic requirement of the mitochondrion for cysteine could be obtained from the cysteine synthesis catalysed by cyanoalanine synthase, but only in the absence of cyanide, unless a more effective substrate than OAS was used in vivo. The situation regarding mitochondrial specific isoenzymes of O-ASTL remains unclear. Three independent reports have shown that putative mitochondrial forms of O-ASTL enzymes were in fact cyanoalanine synthases (Maruyama et al., 2000; Warrilow and Hawkesford, 2000; Hatzfeld et al., 2000). However, it has been shown that a putative Arabidopsis thaliana mitochondrial gene, expressed in E. coli, showed typical characteristics of an O-ASTL enzyme and was not a cyanoalanine synthase (Jost et al., 2000). Therefore, some species seem to possess mitochondrial O-ASTL isoenzymes whilst others do not; the biological significance of these variations needs further investigation. The cyanoalanine synthesis reaction of O-ASTLs A and B is a relatively minor activity compared to the cysteine synthesis reaction, but may be sufficient to convert the cyanide, produced by ethylene biosynthesis, into cyanoalanine. The main functions of O-ASTLs A and B are cysteine synthesis and the removal of excess bisulphide from the cell. The inhibition studies show that significant differences exist between cyanoalanine synthase and O-ASTLs A and B, especially with regard to inhibition by OAS and KCN. Also the differences found between O-ASTLs A
and B, especially inhibition by preincubation by OAS, show that they are different isoenzymes with potentially different bioregulatory functions. Droux et al. established that spinach chloroplast O-ASTL was deactivated by preincubation with OAS in the absence of bisulphide (Droux et al., 1992). The results (Fig. 1) confirm this finding and show that the chloroplastic O-ASTL (B) was more severely deactivated than the cytosolic isoenzyme (A). The cyanoalanine synthase was only slightly inactivated by preincubation with OAS. Further OAS preincubation studies (Fig. 1A) with O-ASTLs A and B show that the inactivation is time-dependent for O-ASTL B, but is less so for O-ASTL A. This suggests that the chloroplastic O-ASTL (B) isoform could be rapidly modulated by the availability of OAS in vivo. The results suggest that for both the cysteine and cyanoalanine synthesis reactions, the general mechanism for the alanyl half-cycle is the same or similar for all three enzymes, with the exception of the binding of OAS to cyanoalanine synthase. The binding of OAS to the active site of cyanoalanine synthase is likely to be sterically hindered because of the bulky O-acetyl group, forcing a different orientation of the OAS molecule relative to that obtained with a chloroalanine molecule. Relatively few changes in the active site, such as the Thr to Met substitution would facilitate such differential binding characteristics. The recent discovery of an allosteric anion binding site on Salmonella O-ASTL (Burkhard et al., 2000) could explain why negative co-operative allosteric kinetics were observed towards bisulphide with spinach O-ASTLs A and B (Warrilow and Hawkesford, 2000). Burkhard et al. discussed the possibility that O-ASTLs are a novel class of b-substituted alanine synthases that are allosterically regulated by anions (Burkhard et al., 2000). Spinach O-ASTLs A and B would be allosterically regulated in vivo by bisulphide anions, whereas cyanoalanine synthase is unlikely to be allosterically regulated. The data presented here (and in Warrilow and Hawkesford, 2000) suggest that the major difference on a mechanistic level, between cyanoalanine synthase and O-ASTLs, lies in the binding of the anion to the enzyme and processing in the anion half-reaction of the enzyme mechanisms. Acknowledgements This project was supported by a 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.
References Bertagnolli BL, Wedding RT. 1977. Purification and initial characterization of different forms of O-acetylserine
Inhibitor studies on O-acetylserine (thiol) lyase and cyanoalanine synthase sulfhydrylase from seedlings of two species of Phaseolus. Plant Physiology 60, 115–121. Bogdanova N, Hell R. 1997. Cysteine synthesis in plants: protein–protein interactions of serine acetyltransferase from Arabidopsis thaliana. The Plant Journal 11, 251–262. Burkhard P, Rao GSJ, Hohenester E, Schnackerz KD, Cook PF, Jansonius JN. 1998. Three-dimensional structure of O-acetylserine sulfhydrylase from Salmonella typhimurium. Journal of Molecular Biology 283, 121–133. Burkhard P, Tai C-H, Ristroph CM, Cook PF, Jansonius JN. 1999. Ligand binding induces a large conformational change in O-acetylserine sulfhydrylase from Salmonella typhimurium. Journal of Molecular Biology 291, 941–953. Burkhard P, Tai C-H, Jansonius JN, Cook PF. 2000. Identification of an allosteric anion-binding site on O-acetylserine sulfhydrylase: structure of the enzyme with chloride bound. Journal of Molecular Biology 303, 279–286. Cook PF, Wedding RT. 1976. A reaction mechanism from steady-state kinetic studies for O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2. Journal of Biological Chemistry 251, 2023–2029. Droux M, Martin J, Sajus P, Douce R. 1992. Purification and characterization of O-acetylserine(thiol)lyase from spinach chloroplasts. Archives of Biochemistry and Biophysics 295, 379–390. Droux M, Ruffet M-L, Douce R, Job D. 1998. Interactions between serine acetyltransferase and O-acetyl-serine(thiol) lyase in higher plants—structural and kinetic properties of the free and bound enzymes. European Journal of Biochemistry 255, 235–245. Gaitonde MK. 1967. A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochemical Journal 104, 627–633. Hasegawa R, Tomoko T, Yuichiro T, Yohji E. 1994. Presence of b-cyanoalanine synthase in unimbibed dry seeds and its activation by ethylene during pre-germination. Physiologia Plantarum 91, 141–146. Hatzfeld Y, Maruyama A, Schmidt A, Noji M, Ishizawa K, Saito K. 2000. b-cyanoalanine synthase is a mitochondrial cysteine synthase-like protein in spinach and Arabidopsis. Plant Physiology 123, 1163–1171. Hendrickson HR, Conn EE. 1969. Cyanide metabolism in higher plants. IV. Purification and properties of the b-cyanoalanine synthase of blue lupine. Journal of Biological Chemistry 244, 2632–2640. Ikegami F, Kaneko M, Kamiyama H, Murakoshi I. 1988b. Purification and characterization of cysteine synthases from Citrullus vulgaris. Phytochemistry 27, 697–701. Ikegami F, Kaneko M, Kobori M, Murakoshi I. 1988a. Purification and characterization cysteine synthase from Brassica juncea. Phytochemistry 27, 3379–3383. Ikegami F, Takayama K, Murakoshi I. 1988d. Purification and properties of b-cyano-L-alanine synthase from Lathyrus latifolius. Phytochemistry 27, 3385–3389. Ikegami F, Takayama K, Tajima C, Murakoshi I. 1988c. Purification and properties of b-cyano-L-alanine synthase from Spinacia oleracea. Phytochemistry 27, 2011–2016. Jost R, Berkowitz O, Wirtz M, Hopkins L, Hawkesford MJ, Hell R. 2000. Genomic and functional characterization of
445
the oas gene family encoding O-acetylserine(thiol)lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana. Gene 253, 237–247. Kuske CR, Ticknor LO, Guzma´n E, Gurley LR, Valdez JG, Thompson ME, Jackson PJ. 1994. Purification and characterization of O-acetylserine sulfhydrylase isoenzymes from Datura innoxia. Journal of Biological Chemistry 269, 6223–6232. Maruyama A, Ishizawa K, Takagi T. 2000. Purification and characterization of b-cyanoalanine synthase and cysteine synthases from potato tubers: are b-cyanoalanine synthase and mitochondrial cysteine synthase same enzyme? Plant and Cell Physiology 42, 200–208. Nakamura T, Yamaguchi Y, Sano H. 1999. Four genes encoding cysteine synthase: isolation and differential responses to sulfur, nitrogen and light. Gene 229, 155–161. Rolland N, Ruffet M-L, Job D, Douce R, Droux M. 1996. Spinach chloroplast O-acetylserine(thiol)lyase exhibits two catalytically non-equivalent pyridoxal-59-phosphate containing active sites. European Journal of Biochemistry 236, 272–282. Saito K, Yokoyama H, Noji M, Murakoshi I. 1995. Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory role in cysteine biosynthesis from watermelon. Journal of Biological Chemistry 270, 16321–16326. Scha¨fer HJ, Haag-Kerwer A, Rausch T. 1998. cDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavy-metal accumulator Brassica juncea L.: evidence for Cd-induction of a putative mitochondrial c-glutamylcysteine synthetase isoform. Plant Molecular Biology 37, 87–97. Segel IH. 1993. Enzyme kinetics: behaviour and analysis of rapid equilibrium and steady-state enzyme systems, 2nd edn. New York: John Wiley and Sons. Smith FW, Hawkesford MJ, Ealing PM, Clarkson DT, Vanden Berg PJ, Belcher AR, Warrilow AGS. 1997. Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter. The Plant Journal 12, 875–884. Tai CH, Nalabolu SR, Jacobson TM, Minter DE, Cook PF. 1993. Kinetic mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2 using the natural and alternative reactions. Biochemistry 32, 6433–6442. Tai CH, Nalabolu SR, Simmons JW, Jacobson TM, Cook PF. 1995. Acid–base chemical mechanism of O-acetylserine sulfhydrylase-A and -B from pH studies. Biochemistry 34, 12311–12322. Warrilow AGS, Hawkesford MJ. 1998. Separation, subcellular location and influence of sulphur nutrition on isoforms of cysteine synthase in spinach. Journal of Experimental Botany 49, 1625–1636. Warrilow AGS, Hawkesford MJ. 2000. Cysteine synthase (O-acetylserine(thiol)lyase) substrate specificities classify the mitochondrial isoform as a cyanoalanine synthase. Journal of Experimental Botany 347, 985–993. Wurtele ES, Nikolau BJ, Conn EE. 1985. Subcellular and developmental distribution of b-cyanoalanine synthase in barley leaves. Plant Physiology 78, 285–290.
