Báo cáo khoa học: Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts doc

Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts Taise Shimaoka 1,2 , Chikahiro Miyake 2 and Akiho Yokota 1,2 1 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, Japan; 2 Research Institute of Innovative Technology for the Earth, Kyoto, Japan Dehydroascorbate reductase (DHAR) reduces dehydro- ascorbate (DHA) to ascorbate with glutathione (GSH) as the electron donor. We analyzed the reaction mechanism of spinach chloroplast DHAR, which had a much higher reaction specificity for DHA than animal enzymes, using a recombinant enzyme expressed in Escherichia coli. Kinetic analysis suggested that the reaction proceeded by a bi-uni-uni-uni-ping-pong mechanism, in which binding of DHA to the free, reduced form of the enzyme was followed by binding of GSH. The K m value for DHA and the summed K m value for GSH were determined to be 53 ± 12 l M and 2.2 ± 1.0 m M , respectively, with a turnover rate of 490 ± 40 s )1 . Incubation of 10 l M DHAR with 1 m M DHA and 10 l M GSH resulted in stable binding of GSH to the enzyme. Bound GSH was released upon reduction of the GSH–enzyme adduct by 2-mercaptoethanol, suggesting that the adduct is a reaction intermediate. Site-directed muta- genesis indicated that C23 in DHAR is indispensable for the reduction of DHA. The mechanism of catalysis of spinach chloroplast DHAR is proposed. Keywords: dehydroascorbate reductase; catalytic mechan- ism; ping-pong mechanism; oxidative stress; ascorbate. Ascorbate functions not only as an antioxidant but also as a substrate for ascorbate peroxidase (APX) and violaxanthin de-epoxidase in chloroplasts [1,2]. APX catalyzes the decomposition of hydrogen peroxide in the active oxygen- scavenging system and the reaction catalyzed by violaxan- thin de-epoxidase in the xanthophyll cycle is involved in the down-regulation of the activity of photosystem II. These enzymes are involved in the dissipation of excess light energy and protect plants from oxidative stress. In reactions catalyzed by APX and violaxanthin de-epoxidase, ascorbate is oxidized to monodehydroascorbate (MDA) and then dehydroascorbate (DHA) is produced via the spontaneous disproportionation of MDA. The regeneration of ascorbate is essential for the maintenance of the activity of the active oxygen-scavenging system and the xanthophyll cycle. MDA and DHA are reduced to ascorbate by MDA reductase and ferredoxin, and DHA reductase (DHAR) in chloroplasts, respectively [3–6]. The reduction of DHA to ascorbate by DHAR (EC 1.8.5.1) involves GSH as the electron donor. Enzymes that reduce DHA are distributed not only in plant cells but also in mammalian cells [7–14]. However, the enzymatic properties of spinach chloroplast DHAR are different from those of other DHA-reducing enzymes. The specific activity of spinach chloroplast DHAR was found to be seven times higher than that of DHAR from rice bran [8,15]. Moreover, spinach chloroplast DHAR has a 100-fold lower K m value for DHA and several-fold higher specific activity than porcine DHAR and other DHA-reducing enzymes [7,11–13,15,16]. DHA-reducing enzymes commonly include a C-X-X-C motif. It has been demonstrated by site-directed muta- genesis that the C22 residue in pig liver thioltransferase is essential for the reduction of DHA [17]. Spinach chloroplast DHAR also has this motif [15] but the highly efficient reduction of DHA by spinach chloroplast DHAR cannot be explained by this motif alone. The difference in k cat between spinach chloroplast DHAR and other DHA- reducing enzymes might be due to differences in mecha- nisms of catalysis. Models for catalysis by DHAR were proposed for pig liver thioltransferase and trypanothione:glutathione disul- fide thioltransferase from Trypanosoma cruzi [18,19]. However, the validity of these models has not been confirmed by kinetics. In the present study, we examined the mechanism of catalysis by spinach chloroplast DHAR. Our kinetic studies showed that catalysis by spinach chloroplast DHAR proceeds by a bi-uni-uni-uni-ping-pong Correspondence to Akiho Yokota, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Fax: + 81 743 72 5569, Tel.: + 81 743 72 5560, E-mail: yokota@bs.aist-nara.ac.jp Abbreviations: APX, ascorbate peroxidase; AsA, ascorbate; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; GSH, glutathione; GSSG, oxidized glutathione; K eq , equilibrium constant; K DHA m , K m for DHA; K GSH1 m , K m for the first-binding molecule of GSH; K GSH2 m , K m for the second-binding molecule of GSH; K DHA i , inhibition constant for DHA; K GSH1 i , inhibition constant for the first-binding molecule of GSH; K GSH2 i , inhibition constant for the second-binding GSH; K AsA m , K m for AsA; K GSSG m , K m for GSSG; K AsA i , inhibition constant for AsA; K GSSG i , inhibition constant for GSSG; MDA, monodehydroascorbate; V max , maximum reaction rate; V maxf , maximum rate of the forward reaction; V maxr , maximum rate of the reverse reaction; V f , forward reaction rate; V r , reverse reaction rate. Enzyme: Dehydroascorbate reductase (EC 1.8.5.1). (Received 11 September 2002, revised 26 December 2002, accepted 7 January 2003) Eur. J. Biochem. 270, 921–928 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03452.x mechanism. A disulfide bond was formed between the enzyme reacted with DHA and GSH, and site-directed mutagenesis revealed that the C23 was essential for DHAR activity. The role of the C residue in the reaction catalyzed by DHAR is discussed. Experimental procedures Materials DHA was purchased from Sigma (St Louis, MO, USA). GSH, GSSG and 2-mercaptoethanol were obtained from Wako Pure Chemical Industries (Osaka, Japan). 4-Fluoro- 7-sulfamoylbenzofurazan was obtained from Dojin (Kuma- moto, Japan). Other chemicals and reagents were of the highest purity commercially available. Assay of the activity of DHAR The reaction rate of DHAR was determined by monitoring the glutathione-dependent production of ascorbate at 265 nm, as described in a previous report [15]. The reaction mixture contained 50 m M potassium phosphate (pH 7.0), 1m M EDTA, DHA and GSH at the indicated concentra- tions, and purified enzyme. Analysis of data All data were fitted to theoretical lines or curves by the least- squares method with the computer program KALEIDAGRAPH 3.08d (Synergy Software, PA, USA). Purification of recombinant DHAR from Escherichia coli E. coli BL21 (DE3) harboring pET3a-DHAR, in which the cDNA for the mature form of spinach chloroplast DHAR had been ligated [15], was grown in 200 mL of LB medium supplemented with 50 lgÆmL )1 ampicillin at 37 °Cfor 12–16 h. All procedures for purification were performed at 0–4 °C. The cultured cells were collected by centrifugation at 4620 g for 10 min and resuspended in 50 mL of 50 m M potassium phosphate (pH 7.8) that contained 1 m M EDTA, 40 m M 2-mercaptoethanol, 1 m M phenylmethylsulfonyl fluoride and 20% (v/v) glycerol. The cells were disrupted with a French Pressure Cell Press (AFPS-20KM; Amicon, MA, USA) at 6895 MPa. The cell extract was centrifuged at 10 000 g for 10 min and the supernatant was used for purification of recombinant DHAR. The supernatant was brought to 40% saturation by addition of (NH 4 ) 2 SO 4 and allowed to stand for 30 min with gentle stirring. After centrifugation at 10 000 g for 10 min, the supernatant was applied to a column (1.6 · 10 cm) of butyl-Toyopearl (Tosoh, Tokyo, Japan), which had been equilibrated with buffer A, which was 50 m M potassium phosphate (pH 7.8) containing 1 m M EDTA, 10 m M 2-mercaptoethanol and 20% (v/v) glycerol. The adsorbed enzyme was eluted with a linear gradient of (NH 4 ) 2 SO 4 (40–0% saturation) in buffer A. The active fractions were appliedtoacolumn(2.6 · 60 cm) of Superdex 75 prep grade (Amersham Pharmacia, Uppsala, Sweden), which had been equilibrated with buffer A containing 0.15 M KCl. The column was developed with the same buffer and the active fractions were pooled and stored at )80 °C. The concentration of purified enzyme was quantitated with a Protein Assay kit from Bio-Rad (Hercules, CA, USA) with bovine serum albumin as the standard. Quantitation of DHAR-bound GSH DHAR in buffer A containing 0.15 M KCl was passed through a column of PD-10 (Amersham Pharmacia), which had been equilibrated with N 2 -purged 50 m M potassium phosphate (pH 7.0) that contained 1 m M EDTA, to remove 2-mercaptoethanol under anaerobic conditions. The reduced-form enzyme (10 l M )wasallowedtoreactwith 1m M DHA and 10 l M GSH at room temperature for 30 s in the presence of 50 m M potassium phosphate (pH 7.0) that contained 1 m M EDTA under N 2 . DHA, GSH and phosphate were removed from the mixture under anaerobic conditions by passage through a column of PD-10 (Amer- sham Pharmacia), which had been equilibrated with 50 m M O 2 -free borate buffer (pH 8.0) that contained 1 m M EDTA. The protein fraction was collected in the small vial, which waspurgedwithN 2 gas through the rubber cap during the collection. Then 100 lL of the enzyme solution were incubated with 50 m M 2-mercaptoethanol at 25 °Cfor 30 min, and evaporated to dryness. The residue was dissolved in 50 lL of distilled water. The 2-mercaptoetha- nol-free solution was mixed with 50 lLof1m M 4-fluoro- 7-sulfamoylbenzofurazan in 0.1 M borate buffer (pH 8.0) and incubated at 50 °C for 5 min. After incubation, the mixture was cooled on ice and acidified by addition of 30 lLof0.1 M HCl. The acidified solution was applied to a column (2.3 · 250 mm) of Wacosil-II 5C18 HG (Wako, Osaka, Japan), which was part of an HPLC system and which had been equilibrated with a mixture of 50 m M potassium hydrogen phthalate (pH 4.0) and acetonitrile (92 : 8, v/v). The column was developed with the same solution at a flow rate of 1.0 mLÆmin )1 . The GSH that had been modified by 4-fluoro-7-sulfamoylbenzofurazan was detected fluorometrically with an excitation at 380 nm and an emission at 510 nm. Site-directed mutagenesis of DHAR Three C residues (C9, C23, C26) in mature DHAR from spinach chloroplasts were individually mutated at one or two positions. The plasmid pET3a-DHAR [15] was digested with SacIandXbaI. The excised DNA fragment containing the cDNA for part of the mature form of spinach chloroplast DHAR was inserted at the SacI–XbaIsitein pUC18. Amplification by PCR was carried out with the pUC18 vector that contained the fragment as template and the following primers: for C9S, P2 and P3; for C23S, P1 and P4; for C26S, P1 and P5; and for C9S/C26S, P1, P2, P3 and P5. The oligonucleotide primers sequences used in PCR were as follows: P1, 5¢-AGCTTGTTGGGGGTGGT GAC-3¢;P2,5¢-AATCTGTCACCACCCCCAAC-3¢;P3, 5¢-CCTTGACG GATATTTGGAGTG-3¢;P4,5¢-TGGCG ATT CTCCATTTTGCCAAAGAGTG-3¢;andP5,5¢-TG GCGATTGTCCATTTT CCCAAAGAGTG-3¢.Mutated bases are underlined. The PCR products were phosphoryl- ated and self-ligated. After mutation of DHAR, the 922 T. Shimaoka et al.(Eur. J. Biochem. 270) Ó FEBS 2003 sequences of the DNA fragments were confirmed by nucleotide sequencing with vector primers, M13 reverse and forward primers, and a Thermo Sequenase II dye terminator cycle-sequencing Premix kit (Amersham Phar- macia) with an automated DNA sequencer (model 373; Applied Biosystems, CA, USA). The mutated DNA fragments were used individually to replace the SacI-XbaI fragment of pET3a-DHAR. Proteins encoded by the vectors that included cDNAs for mutant DHARs, namely pDHAR-C9S, pDHAR-C23S, pDHAR-C26S and pDHAR-C9S/C26S, were expressed in E. coli BL21 (DE3). Recombinant enzymes were purified as described above. SDS/PAGE SDS/PAGE was performed with 12.5% polyacrylamide gels as described by Laemmli [20]. Proteins on the gels were stained with Coomassie Brilliant Blue R-250 (Nacalai tesque, Kyoto, Japan). Western blotting After separation by SDS/PAGE of each DHAR, the protein on the gel was transferred to a poly(vinylidene difluoride) membrane by a semidry blotting method. A specific antibody, raised in rabbit against recombinant spinach chloroplast DHAR, was used at a dilution of 1/2000 in 30 m M Tris/HCl (pH 7.5) that contained 200 m M NaCl and 5% (w/v) skim milk. Immunoreactive proteins, which bound with antibodies against rabbit IgG that had been conjugated to horseradish peroxidase, on the poly(vinylidene difluoride) membrane were revealed with an Immunostaining HRP-1000 kit (Konica, Tokyo, Japan) according to the manufacturer’s instructions. Results and discussion Initial velocity and product inhibition of the reaction catalyzed by spinach chloroplast DHAR We purified recombinant DHAR to homogeneity from E. coli that expressed a cDNA for mature DHAR from spinach chloroplasts (Fig. 1). Because the K m values for DHA and GSH, and k cat of the recombinant DHAR were the same as those of the DHAR purified from fresh leaves of spinach [15], we used the recombinant DHAR in this study. DHAR catalyzes the reduction of DHA to ascorbate with GSH as the electron donor [21,22], as follows: DHA þ 2GSH ! Ascorbate þ GSSG Thus, the reduction of DHA by DHAR is a ter-bi reaction. We measured the initial velocity of the reaction catalyzed by DHAR with the concentration of GSH fixed at 0.2, 0.3, 0.5, 0.8, 1.0, 2.0 or 4.0 m M , varying the concentration of DHA from 0.02 to 0.5 m M . We also measured the activity when we varied the concentration of GSH from 0.2 to 4.0 m M with the concentration of DHA fixed at 0.02, 0.03, 0.05, 0.07, 0.1, 0.2 or 0.5 m M . Double-reciprocal plots for activity vs. various concentrations of one substrate yielded straight lines at the various fixed concentrations of the other substrate (Fig. 2A,B). The lines crossed in the second quadrant. The velocity of the reaction catalyzed by DHAR in the absence of reaction products can be expressed as Eqn (1) because the DHAR reaction is a ter bi reaction: v ¼ V max ½DHA½GSH 2 =fð½DHAÀ½GSHÞ ð1Þ The numerator of Eqn (1) is represented by the product of the maximum velocity (V max ) and the concentrations of substrates, and the denominator by the function of the concentrations of substrates. The straight lines of the double-reciprocal plots in Fig. 2 indicate that the denomi- nator of Eqn (1) for the DHAR-catalyzed reaction does not include a constant, and show that the DHAR-catalyzed reaction proceeds via a ping-pong mechanism. Two mech- anisms have been proposed for ping-pong-ter-bi reactions: bi-uni-uni-uni-ping-pong and uni-uni-bi-uni-ping-pong [23]. By replotting the slopes and the intercepts with the y-axis of the lines in Fig. 2 against the reciprocals of the concentrations of the substrates (Fig. 2, insets a–d), straight lines are obtained. In the case of ping-pong mechanisms, Eqn (1) is transformed as follows: v ¼ V max  f 1 ð½GSHÞ Â ½DHA=f½DHAþf 2 ð½GSHÞg ð2AÞ ¼ V max  f 1 ð½DHAÞ Â ½GSH=f½GSHþf 2 ð½DHAÞg ð2BÞ Fig. 1. SDS/PAGE and Western blotting analysis of purified wild type and mutant forms of DHAR. Recombinant wild type and mutant forms of DHAR were produced in E. coli and purified as described in ÔExperimental proceduresÕ. The purified wild type and mutant enzymes were subjected to SDS/PAGE on a 12.5% polyacrylamide gel. The proteins on the gel were stained with Coomassie Brilliant Blue R-250 (lanes 1–5) or transferred to a poly(vinylidene difluoride) membrane for Western blotting analysis (lanes 6–10). Each lane was loaded with 2 lg of enzyme for staining with Coomassie Brilliant Blue and 10 ng of protein for Western blotting. M, Molecular mass markers; lanes 1 and 6, wild type DHAR; lanes 2 and 7, C9S DHAR; lanes 3 and 8, C23S DHAR; lanes 4 and 9, C26S DHAR; and lanes 5 and 10, C9S/C26S DHAR. The following proteins were used as molecular mass markers: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbu- min (43 kDa), carbonic anhydrase b (30 kDa), trypsin inhibitor (20.1 kDa), and a-lactalbumin (14.4 kDa). Ó FEBS 2003 Catalytic mechanism of chloroplast DHAR reaction (Eur. J. Biochem. 270) 923 The straight lines in insets a–d of Fig. 2 indicate that both 1/f 1 ([GSH]) and f 2 ([GSH])/f 1 ([GSH]) are represented by the linear function of the reciprocals of the concentrations of GSH and, moreover, that both 1/f 1 ([DHA]) and f 2 ([DHA])/ f 1 ([DHA]) are represented by the linear function of the reciprocals of the concentrations of DHA. Therefore, it appears that the reaction catalyzed by DHAR proceeds via a bi-uni-uni-uni-ping-pong mechanism and that the sub- strate that binds last is GSH. The last-binding substrate of GSH means that the first product is ascorbate and that GSSG is the final product in the catalytic cycle of the DHAR reaction, as GSSG is produced from two molecules of GSH. We performed product-inhibition studies to confirm the order of binding of DHA and GSH to DHAR. Double- reciprocal plots of reaction velocity against the concentra- tion of DHA in the presence of 4.0 m M GSH and 0, 2.0 or 5.0 m M GSSG gave straight lines that pivoted counter- clockwise on the point at which the lines intersected (Fig. 3A). Double-reciprocal plots of velocity vs. the concentration of GSH in the presence of 0.5 m M DHA and 0, 2.0 or 5.0 m M GSSG yielded parabolic curves (Fig. 3B). These results indicate that GSSG acts as a competitive inhibitor with respect to DHA and as a mixed- type inhibitor with respect to GSH. The competitive inhibition by GSSG with respect to DHA is consistent with a mechanism in which DHA binds first to the enzyme (Fig. 4). The activity of spinach chloroplast DHAR was inhibited by incubation with iodoacetic acid and such inhibition was suppressed by the addition of DHA [15], suggesting that a C residue of DHAR might interact with DHA. In the chemical reaction between DHA and GSH, glutathionyl Fig. 3. Inhibition of the DHAR-catalyzed reaction by GSSG at various concentrations of DHA (A) and GSH (B). The concentrations of GSH and DHA were 4.0 m M in (A) and 0.5 m M in (B). The concentrations of GSSG were 0 m M (d), 2.0 m M (m)and4.0m M (j). Each reaction mixture contained 20 ng of enzyme. For other details, see Experi- mental procedures. Fig. 2. Double-reciprocal plots of the initial velocity vs. the concentra- tion of one substrate at various fixed concentrations of the other sub- strate. (A) Reciprocals of initial rates of reduction of DHA are plotted against the reciprocals of concentrations of DHA at several fixed concentrations of GSH. The concentrations of GSH were 0.3 m M (d), 0.5 m M (j), 0.8 m M (m), 1.0 m M (s), 2.0 m M (h), 3.0 m M (n)and 4.0 m M (e). In insets (a) and (b), the slope and the intercept on the y-axis, respectively, are replotted against the reciprocals of the con- centrations of GSH. (B) Reciprocals of initial rates of reduction of DHA were plotted against the reciprocals of concentrations of GSH at fixed concentrations of DHA. The concentrations of DHA were 0.02 m M (d), 0.03 m M (j), 0.05 m M (m), 0.07 m M (s), 0.1 m M (h), 0.2 m M (n)and0.5m M (e). In insets (c) and (d), the slope and the intercept on the y-axis, respectively, are replotted against the recipro- cals of the concentrations of DHA. Each reaction mixture contained 20 ng of enzyme. For other details, see Experimental procedures. 924 T. Shimaoka et al.(Eur. J. Biochem. 270) Ó FEBS 2003 hemiketal is formed first as a reaction intermediate [24]. Therefore, it is likely that a cysteinyl-thiohemiketal complex is formed between DHA and the sulfhydryl group of a C residue of DHAR in its reduced form. The most plausible reaction mechanism that incorporates our steady-state kinetic studies seems to be the bi-uni-uni- uni-ping-pong mechanism (Fig. 4). The sulfhydryl group of a C residue in the reduced enzyme, E-S – , reacts with DHA. The reducing equivalents of the sulfhydryl groups of the C residue reduce DHA to ascorbate, and generate the oxidized form of the enzyme, E-S-SG. A second molecule of GSH then reduces E-S-SG to generate E-S – and GSSG. We calculated the kinetic parameters, as described in the Appendix, and summarized in Table 1. The calculated K GSH m was the sum of the values of K m for the first-binding molecule of GSH and the second-binding molecule of GSH since we were unable to calculate separately K GSH1 m and K GSH2 m . The mechanism of catalysis suggests that the differences in specific activity between spinach chloroplast DHAR and other DHA-reducing proteins, such as the thioltransferase from pig liver and trypanothione:glutathione disulfide thioltransferase from T. cruzi, might be due to differences in the mechanisms of catalysis. The mechanism proposed for T. cruzi enzyme [19] includes the formation of gluta- thionyl-thiohemiketal by DHA and GSH [24] on the enzyme. Our steady-state kinetic studies for spinach chloroplast DHAR do not support such a mechanism (Figs 2 and 3). In contrast, the reduction of DHA to ascorbate by C23S DHAR had the DHAR activity similar to that of the T. cruzi enzyme, and may proceed via the reaction mechanism of the T. cruzi enzyme. Two mechanisms were proposed for the pig liver enzyme [18]. One is the same as that for spinach chloroplast DHAR. However, it is not clear, because neither the steady-state kinetics nor the structure of the reaction intermediate has been examined with the pig liver enzyme. In the other mechanism, an intramolecular disulfide bond was proposed to be formed in the enzyme during the DHA-reducing reaction. However, mutation of C9 and C26 to S residues results in the appearance of all DHAR activity in the present study (Table 1). Detection of the oxidized form enzyme, E-S-SG To detect E-S-SG, we performed the following experiment. We reacted E-S – with an excess of DHA to generate a cysteinyl-thiohemiketal complex, E-S-DHA. Then, we incubated E-S-DHA in 2-mercaptoethanol-free medium with equimolar GSH to that of the enzyme. E-S-SG was freed of residual DHA and GSH by gel filtration and then excess 2-mercaptoethanol was added to E-S-SG to reduce the disulfide bond that had formed between the enzyme and GSH. The GSH released from E-S-SG was detected by HPLC as described in Experimental procedures. When E-S – was reacted with excess DHA, we detected GSH with a retention time of 7.4 min (Fig. 5). The detected GSH was 5.8% of the reacted enzyme when we quantified them by the standard addition method. No GSH was detected at this retentiontimewhentheenzymewasreactedwithGSHonly (Fig. 5). The results indicate that the reduced form of DHAR reacted first with DHA and then E-S-DHA reacted with GSH to generate E-S-SG. The low yield of E-S-SG might be due to the higher rate of the reaction between E-S-SG and the second GSH than that of the reaction between E-S-DHA and the first GSH. Identification of the C residue involved in the reaction catalyzed by chloroplast DHAR Spinach chloroplast DHAR contains three C residues, namely C9, C23 and C26. C9 and C23 are conserved in plant DHARs. C26 is conserved in spinach chloroplast and in Arabidopsis DHARs but is replaced to the S residue in rice bran DHAR (Fig. 6). We purified the mutated DHARs that had been expressed in E. coli.SDS/PAGErevealedthat each enzyme had been purified to homogeneity, and the purified enzymes were confirmed to be forms of DHAR by Western blotting with antibodies specific for spinach chloroplast DHAR (Fig. 1). The kinetic parameters of wild type and mutant DHARs were 1 described as above. C23S DHAR had almost no activity (Table 1). The k cat of C26Swashalfthatofthewild-typeDHAR,whilethek cat of Fig. 4. The most plausible mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts. E-S – and E-S-S-G mean the reduced form and the oxidized form enzymes, respectively [19]. Table 1. Kinetic parameters of wild-type and mutant forms of DHAR from spinach chloroplasts. Values of k cat were calculated using a molecular mass of 24 kDa. Values of k cat and K m are given as means ±SD (n ¼ 3–5). k cat (s )1 ) K m for DHA (l M ) K m for GSH (m M ) k cat =K DHA m ( M )1 Æs )1 ) k cat =K GSH m ( M )1 Æs )1 ) Wild-type 490 ± 40 53 ± 12 1.1 ± 0.5 (9.2 ± 2.1)Æ10 6 (5.2 ± 1.9)Æ10 5 C9S 420 ± 30 19 ± 14 0.95 ± 0.07 (3.1 ± 2.4)Æ10 7 (4.5 ± 0.6)Æ10 5 C23S <1 – – – – C26S 280 ± 30 26 ± 6 0.69 ± 0.2 (1.1 ± 0.3)Æ10 7 (4.2 ± 0.9)Æ10 5 C9/26S 210 ± 10 58 ± 7 1.1 ± 0.3 (3.7 ± 0.3)Æ10 6 (2.0 ± 0.5)Æ10 5 Ó FEBS 2003 Catalytic mechanism of chloroplast DHAR reaction (Eur. J. Biochem. 270) 925 C9S was slightly lower than that of wild-type DHAR. The k cat of C9S/C26S was 210 ± 10 s )1 . The respective K m values for the two substrates of wild type C9S, C26S and C9S/C26S DHARs, were almost identical. These results indicate that C23 is essential for spinach chloroplast DHAR to have the high specific activity and suggest that this residue may be involved in the formation of the disulfide bond with GSH in the cysteinyl-thiohemiketal. Other C residues might also be involved in the reaction of the spinach enzyme, but their contributions were not significant (Table 1). Influence of reaction products on the activity of DHAR In a previous paper [15], we discussed the ability of spinach chloroplast DHAR to function as an ascorbate-regener- ating enzyme in vivo. In the present study, we analyzed the effects of product-inhibition on the activity of DHAR to clarify the possible effects of reaction products on the activity of DHAR in vivo. Spinach chloroplasts contain 12–25 m M ascorbate [25,26] and 3–4 m M glutathione [26,27]. More than 90% of the ascorbate and a similar percentage of glutathione are found in the reduced forms under nonstress conditions. Thus, the concentrations of DHA and GSSG might be 2.5 m M and 0.4 m M at maximum, respectively. When we assayed the activity of spinach chloroplast DHAR in the presence of 4.0 m M GSH and 0.5 m M DHA, the activity decreased by 75% upon addition of 2.0 m M GSSG, a concentration of GSSG that is much higher than that in chloroplasts (Fig. 3). Considering this result, we can speculate that the inhibition of DHAR activity by GSSG might not affect the activity of spinach chloroplast DHAR in vivo, under conditions where the concentrations of GSH and GSSG are 4 m M and 0.4 m M , respectively. By contrast, when we assayed the activity of chloroplast DHAR in the presence of 1.0 m M GSH and 0.1 m M DHA, the activity decreased by 40% upon addition of 20 m M ascorbate (T. Shimaoka, C. Miyake & A. Yokota, unpublished results). This finding suggests that ascorbate lowers the activity of DHAR in spinach chloroplasts, in which the concentrations of ascorbate and DHA are 25 m M and 2.5 m M , respectively. In our earlier estimate of the rate of formation of superoxide at photosystem I [28], we proposed that MDA would be formed at a rate of 300 lmolÆmg chlorophyll )1 Æh )1 in the reaction catalyzed by APX for decomposition of hydrogen peroxide at a light intensity of 1400 lmol photonsÆm )2 Æs )1 in air. Most of the MDA formed in the water-water cycle is directly reduced to ascorbate by ferredoxin [1]. If 10% of the MDA were disproportionated to ascorbate and DHA, the rate of formationofDHAwouldbe15 lmolÆmg chlorophyll )1 Æh )1 . This rate corresponds closely to 20% of the maximum activity of chloroplast DHAR that we measured in our previous study [15]. Therefore, it appears that DHAR can reduce all available DHA to ascorbate under nonstress conditions, even if the maximum activity of DHAR is inhibited by 40% by ascorbate, the concentration of which might range from 12 to 25 m M . However, we cannot ignore the possibility that the activity of DHAR, in terms of the regeneration of ascorbate, might be limited under stress conditions, where the rate of production of DHA is elevated. Acknowledgements This study was partly supported by the Petroleum Energy Center and the Research Association for Biotechnology subsidized by the Ministry of Economy, Trade and Industry of Japan. References 1. Asada, K. (1999) The water-water cycle in chloroplasts: scaven- ging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 601–639. 2. Niyogi, K.K. (1999) Photoprotection revisited: genetic and molecular approaches. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 333–359. 3. Jablonski, P.P. & Anderson, J.W. (1981) Light-dependent reduc- tion of dehydroascorbate by ruptured pea chloroplasts. Plant Physiol. 67, 1239–1244. 4. Nakano, Y. & Asada, K. (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. 5. Hossain, M.A., Nakano, Y. & Asada, K. (1984) Mono- dehydroascorbate reductase in spinach chloroplasts and its parti- cipation in regeneration of ascorbate for scavenging hydrogen peroxide. Plant Cell Physiol. 25, 385–395. Fig. 6. The amino acid sequences around the C-X-X-C motif in spinach chloroplast DHAR and DHA-reducing enzymes from other organisms. White letters in black boxes represent C residues of the following sequences: spinach chloroplast DHAR [15], Arabidopsis chloroplast DHAR [15], rice bran DHAR [30], bovine protein disulphide iso- merase (PDI) [31], T. cruzi trypanothione-glutathione thioltransferase (p52) [32], rat liver DHAR [33], rice glutaredoxin (Grx) [12], and pig liver thioltransferase [34]. Fig. 5. Detection of DHAR-bound GSH by HPLC. The 2-mercapto- ethanol-reduced enzyme (10 l M ), from which 2-mercaptoethanol had been removed under anaerobic conditions was reacted with 1 m M DHA and 10 l M GSH (solid line) or 10 l M GSH (dashed line) at room temperature for 30 s in 50 m M potassium phosphate (pH 7.0) that contained 1 m M EDTA under aerobic conditions. For other details 2 , see Experimental procedures. 926 T. Shimaoka et al.(Eur. J. Biochem. 270) Ó FEBS 2003 6. Miyake, C. & Asada, K. (1994) Ferredoxin-dependent photo- reduction of the monodehydroascorbate radical in spinach thylakoids. Plant Cell Physiol. 35, 539–549. 7. Wells, W.W., Xu, D.P., Yang, Y. & Rocque, P.A. (1990) Mam- malian thioltransferase (glutaredoxin) and protein disulfide iso- merase have dehydroascorbate reductase activity. J. Biol. Chem. 265, 15351–15364. 8. Kato, Y., Urano, J., Maki, Y. & Ushimaru, T. (1997) Purification and characterization of dehydroascorbate reductase from rice. Plant Cell Physiol. 38, 173–178. 9. Hossain, M.A. & Asada, K. (1984) Purification of dehydro- ascorbate reductase from spinach and its characterization as a thiol enzyme. Plant Cell Physiol. 25, 85–92. 10. Dipierro, S. & Borraccino, G. (1991) Dehydroascorbate reductase from potato tubers. Phytochemistry 30, 427–429. 11. Xu, D.P., Washburn, M.P., Sun, G.P. & Wells, W.W. (1996) Purification and characterization of a glutathione dependent dehydroascorbate reductase from human erythrocytes. Biochem. Biophys. Res. Commun. 221, 117–121. 12. Sha, S., Minakuchi, K., Higaki, N., Sato, K., Ohtsuki, K., Kurata, A., Yoshikawa, H., Kotaru, M., Masumura, T., Ichihara, K. & Tanaka, K. (1997) Purification and characterization of gluta- redoxin (thioltransferase) from rice (Oryza sativa L.). J. Biochem. (Tokyo) 121, 842–848. 13. Washburn, M.P. & Wells, W.W. (1999) Identification of the dehydroascorbic acid reductase and thioltransferase (Gluta- redoxin) activities of bovine erythrocyte glutathione peroxidase. Biochem. Biophys. Res. Commun. 257, 567–571. 14. Vethanayagam, J.G., Green, E.H., Rose, R.C. & Bode, A.M. (1999) Glutathione-dependent ascorbate recycling activity of rat serum albumin. Free Rad. Biol. Med. 26, 1591–1598. 15. Shimaoka, T., Yokota, A. & Miyake, C. (2000) Purification and characterization of chloroplast dehydroascorbate reductase from spinach leaves. Plant Cell Physiol. 41, 1110–1118. 16. Maellaro, E., Del Bello, B., Sugherini, L., Comporti, M. & Casini, A.F. (1997) Purification and characterization of glutathione- dependent dehydroascorbate reductase from rat liver. Methods Enzymol. 279, 30–35. 17. Yang, Y. & Wells, W.W. (1991) Identification and characteriza- tion of the functional amino-acids at the active center of pig liver thioltrasferase by site-directed mutagenesis. J. Biol. Chem. 266, 12759–12765. 18. Washburn, M.P. & Wells, W.W. (1999) The catalytic mechanism of the glutathione-dependent dehydroascorbate reductase activity of thioltransferase (glutaredoxin). Biochemistry 38, 268–274. 19. Moutiez, M., Quemeneur, E., Sergheraert, C., Lucas, V., Tartar, A. & Davioud-Charvet, E. (1997) Glutathione-dependent activities of Trypanosoma cruzi p52makesitanewmemberof the thiol:disulphide oxidoreductase family. Biochem. J. 322, 43–48. 20. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacterophage T4. Nature 227, 680–685. 21. Mapson, L.W. (1958) Metabolism of ascorbic acid in plants: function. Annu. Rev. Plant Physiol. 9, 119–150. 22. Foyer, C.H. & Halliwell, B. (1977) Purification and properties of dehydroascorbate reductase from spinach leaves. Phytochemistry 16, 1347–1350. 23. Segel, I.H. (1975) Enzymatic Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley, New York, USA. 24. Winkler, B.S., Orselli, S.M. & Rex, T.S. (1994) The redox couple between glutathione and ascorbic acid: a chemical and physiolo- gical perspective. Free Rad. Biol. Med. 17, 333–349. 25. Foyer, C., Rowell, J. & Walker, D. (1983) Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination. Planta 157, 239–244. 26. Law, M.Y., Charles, S.A. & Halliwell, B. (1983) Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts. Biochem. J. 210, 899–903. 27. Foyer, C.H. & Halliwell, B. (1976) The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133, 21–25. 28. Miyake, C. & Yokota, A. (2000) Determination of the rate of photoreduction of O 2 in the water-water cycle in watermelon leaves and enhancement of the rate by limitation of photosynth- esis. Plant Cell Physiol. 41, 335–343. 29. King, E.L. & Altman, C. (1956) A schematic method of deriving the rate laws for enzyme-catalyzed reactions. J. Phys. Chem. 60, 1375–1378. 30. Urano, J., Nakagawa, T., Maki, Y., Masumura, T., Tanaka, K., Murata, N. & Ushimaru, T. (2000) Molecular cloning and char- acterization of a rice dehydroascorbate reductase. FEBS Lett. 466, 107–111. 31. Yamauchi, K., Yamamoto, T., Hayashi, H., Koya, S., Takikawa, H., Toyoshima, K. & Horiuchi, R. (1987) Sequence of membrane- associated thyroid hormone binding protein from bovine liver: Its identity with protein disulphide isomerase. Biochem. Biophys. Res. Commun. 146, 1485–1492. 32. Schoneck, R., Plumas-Marty, B., Taibi, A., Billaut-Mulot, O., Loyens,M.,Gras-Masse,H.,Capron,A.&Ouaissi,A.(1994) Trypanosoma cruzi cDNA encodes a tandemly repeated domain structure characteristic of small stress proteins and glutathione S-transferase. Biol. Cell 80, 1–10. 33. Ishikawa, T., Casini, A.F. & Nishikimi, M. (1998) Molecular cloning and functional expression of rat liver glutathione-depen- dent dehydroascorbate reductase. J. Biol. Chem. 273, 28708– 28712. 34. Yang, Y., Gan, Z R. & Wells, W.W. (1989) Cloning and sequencing the cDNA encoding pig liver thioltransferase. Gene 83, 339–346. Appendix Mathematical representations of the kinetic model According to the King–Altman method [29], the reaction scheme can be drawn as Fig. 4. The complete rate equation for the bi uni uni uni ping pong is obtained from the above five, four-sided King–Altman interconversion patterns is: in the absence of reaction products, m V max ¼ ½DHA½GSH 2 K DHA i K GSH1 m ½GSHþðK GSH1 m þ K GSH2 m Þ½DHA½GSHþK DHA m ½GSH 2 þ½DHA½GSH 2 ðA1Þ Ó FEBS 2003 Catalytic mechanism of chloroplast DHAR reaction (Eur. J. Biochem. 270) 927 and in the presence of reaction products, m ¼ V maxf V maxr ½DHA½GSH 2 À AsA ½ GSSG ½ K eq  V r K DHA i K GSH1 m ½GSHþV r K GSH2 m ½DHA½GSHþV r K GSH1 m ½DHA½GSH þV r K DHA m ½GSH 2 þ V r ½GSH 2 þ V f K GSSG m ½DHA½AsA K DHA i K eq þ V f K GSSG m ½DHA½GSH½AsA K DHA i K GSH1 i K eq þ V f K GSSG m ½AsA K eq þ V f K AsA m ½GSSG K eq þ V f ½AsA½GSSG K eq þ V r K DHA m K GSH2 i ½GSH½GSSG þ V r K DHA i K GSH1 i ½GSH½GSSG K GSSG i þ V r K DHA m K GSH2 i ½GSH½AsA½GSSG K AsA i K GSSG i þ V r K DHA m ½GSH 2 ½GSSG K GSSG i ðA2Þ Determination of kinetic parameters We determined Michaelis constants and k cat from our initial-velocity experiments using Eqn (A2). We can trans- form Eqn (A2) as follows: v ¼ V max ½GSH ½GSHþK GSH1 m þ K GSH2 m ½DHA ½DHAþ K DHA i K GSH1 m þ K DHA m ½GSH K GSH1 m þ K GSH2 m ½GSH ðA3Þ The plots of initial velocity at various fixed concentrations of GSH and varying concentrations of DHA were fitted to the Michaelis–Menten equation 3 v ¼ m 1 · [DHA]/ ([DHA] + m 2 ). Calculations for m 1 and m 2 were made by application of the computer program KALEIDAGRAPH 3.08d, where m 1 and m 2 represent V max ½GSH=ð½GSHþ K GSH1 m þ K GSH2 m Þ and ðK DHA i K GSH1 m þ K DHA m ½GSHÞ= ðK GSH1 m þ K GSH2 m þ½GSHÞ, respectively. To determine V max , K GSH1 m þ K GSH2 m , K DHA i and K DHA m , we generated double-reciprocal plots between m 1 and the concentration of GSH, and double-reciprocal plots between m 1 /m 2 and the concentration of GSH. The slope and intercept of the former plot gave V max and K GSH1 m þ K GSH2 m while the slope and intercept of the latter plot gave K DHA i and K DHA m . 2 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 4 3 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 5 928 T. Shimaoka et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . k cat of C26Swashalfthatofthewild-typeDHAR,whilethek cat of Fig. 4. The most plausible mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts. E-S – and. at the various fixed concentrations of the other substrate (Fig. 2A,B). The lines crossed in the second quadrant. The velocity of the reaction catalyzed by