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 Shimaoka1,2, Chikahiro Miyake2and Akiho Yokota1,2 1 Graduate School of Biological Scienc

Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts

Taise Shimaoka1,2, Chikahiro Miyake2and Akiho Yokota1,2

1

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, Japan;2Research Institute of Innovative Technology for the Earth, Kyoto, Japan

Dehydroascorbate reductase (DHAR) reduces

dehydro-ascorbate (DHA) to dehydro-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 formof the enzyme was followed

by binding of GSH The Kmvalue for DHA and the summed

Kmvalue for GSH were determined to be 53 ± 12 lMand

2.2 ± 1.0 mM, respectively, with a turnover rate of

490 ± 40 s)1 Incubation of 10 lM DHAR with 1 mM DHA and 10 lMGSH 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 mechanmechan-ism; 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 systemand the reaction catalyzed by

violaxan-thin de-epoxidase in the xanthophyll cycle is involved in the

down-regulation of the activity of photosystemII These

enzymes are involved in the dissipation of excess light energy

and protect plants fromoxidative 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 systemand 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 fromrice bran [8,15] Moreover, spinach chloroplast DHAR has a 100-fold lower Kmvalue 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 kcat 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 , equilibriumconstant;

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)

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 fromSigma (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 mM potassiumphosphate (pH 7.0),

1 mMEDTA, 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 programKALEIDAGRAPH

3.08d (Synergy Software, PA, USA)

Purification of recombinant DHAR fromEscherichia coli

E coliBL21 (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 37C for

12–16 h

All procedures for purification were performed at 0–4C

The cultured cells were collected by centrifugation at 4620 g

for 10 min and resuspended in 50 mL of 50 mMpotassium

phosphate (pH 7.8) that contained 1 mM EDTA, 40 mM

2-mercaptoethanol, 1 mM 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 (NH4)2SO4and 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 mM potassium

phosphate (pH 7.8) containing 1 mM EDTA, 10 mM

2-mercaptoethanol and 20% (v/v) glycerol The adsorbed

enzyme was eluted with a linear gradient of (NH4)2SO4

(40–0% saturation) in buffer A The active fractions were

applied to a colum n (2.6· 60 cm) of Superdex 75 prep grade

(Amersham Pharmacia, Uppsala, Sweden), which had been

equilibrated with buffer A containing 0.15 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 fromBio-Rad (Hercules, CA, USA) with bovine serumalbumin as the standard Quantitation of DHAR-bound GSH

DHAR in buffer A containing 0.15M KCl was passed through a column of PD-10 (Amersham Pharmacia), which had been equilibrated with N2-purged 50 mM potassium phosphate (pH 7.0) that contained 1 mMEDTA, to remove 2-mercaptoethanol under anaerobic conditions The reduced-formenzyme (10 lM) was allowed to react with

1 mMDHA and 10 lMGSH at roomtemperature for 30 s

in the presence of 50 mM potassiumphosphate (pH 7.0) that contained 1 mM EDTA under N2 DHA, GSH and phosphate were removed from the mixture under anaerobic conditions by passage through a column of PD-10 (Amer-shamPharmacia), which had been equilibrated with 50 mM

O2-free borate buffer (pH 8.0) that contained 1 mMEDTA The protein fraction was collected in the small vial, which was purged with N2gas through the rubber cap during the collection Then 100 lL of the enzyme solution were incubated with 50 mM 2-mercaptoethanol at 25C for

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 lL of 1 mM 4-fluoro-7-sulfamoylbenzofurazan in 0.1M borate buffer (pH 8.0) and incubated at 50C for 5 min After incubation, the mixture was cooled on ice and acidified by addition of

30 lL of 0.1MHCl 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 systemand which had been equilibrated with a mixture of 50 mM potassiumhydrogen 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 SacI and XbaI The excised DNA fragment containing the cDNA for part of the mature form of spinach chloroplast DHAR was inserted at the SacI–XbaI site in 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¢-CCTTGACGGATATTTGGAGTG-3¢; P4, 5¢-TGGCG ATTCTCCATTTTGCCAAAGAGTG-3¢; and P5, 5¢-TG GCGATTGTCCATTTTCCCAAAGAGTG-3¢ Mutated bases are underlined The PCR products were phosphoryl-ated and self-ligphosphoryl-ated After mutation of DHAR, the

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 mMTris/HCl (pH 7.5) that contained 200 mM

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 manufacturerỖs 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 Km values for

DHA and GSH, and kcatof 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:

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 mM, varying the concentration of

DHA from0.02 to 0.5 mM We also measured the activity

when we varied the concentration of GSH from0.2 to

4.0 mMwith the concentration of DHA fixed at 0.02, 0.03,

0.05, 0.07, 0.1, 0.2 or 0.5 mM 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 Ử Vmax  ơDHAơGSH2=fđơDHA  ơGSHỡ đ1ỡ The numerator of Eqn (1) is represented by the product of the maximum velocity (Vmax) 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 aỜd), straight lines are obtained In the case of ping-pong mechanisms, Eqn (1) is transformed as follows:

v Ử Vmax f1đơGSHỡ  ơDHA=fơDHA ợ f2đơGSHỡg

đ2Aỡ

Ử Vmax f1đơDHAỡ  ơGSH=fơGSH ợ f2đơDHAỡg

đ2Bỡ

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

Experimental procedures 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 1Ờ5) or transferred to a poly(vinylidene difluoride) membrane for Western blotting analysis (lanes 6Ờ10) 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 serumalbumin (67 kDa), ovalbu-min (43 kDa), carbonic anhydrase b (30 kDa), trypsin inhibitor (20.1 kDa), and a-lactalbumin (14.4 kDa).

The straight lines in insets a–d of Fig 2 indicate that both

1/f1([GSH]) and f2([GSH])/f1([GSH]) are represented by the

linear function of the reciprocals of the concentrations of

GSH and, moreover, that both 1/f1([DHA]) and f2([DHA])/

f1([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 subsub-strate 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 fromtwo 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 mMGSH and 0, 2.0 or 5.0 mM 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 mM DHA and 0, 2.0 or 5.0 mM 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) and 4.0 m 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 concentraconcentra-tions 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) and 0.5 m 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.

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

formof 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 KGSH

m was the sumof the values of Kmfor the first-binding molecule

of GSH and the second-binding molecule of GSH since

we were unable to calculate separately KGSH1

m and KGSH2

The mechanism of catalysis suggests that the differences

in specific activity between spinach chloroplast DHAR and

other DHA-reducing proteins, such as the thioltransferase

frompig 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 fromE-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 retention tim e when the enzym e was reacted with GSH only (Fig 5) The results indicate that the reduced formof 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/PAGE revealed that 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

C23S DHAR had almost no activity (Table 1) The kcatof C26S was half that of the wild-type DHAR, while the kcatof

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 ) kcat=K DHA

m ( M )1 Æs)1) kcat=K GSH

m ( M )1 Æs)1) Wild-type 490 ± 40 53 ± 12 1.1 ± 0.5 (9.2 ± 2.1)Æ106 (5.2 ± 1.9)Æ105

C9S was slightly lower than that of wild-type DHAR The

kcat of C9S/C26S was 210 ± 10 s)1 The respective Km

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 mM ascorbate [25,26] and 3–4 mM 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 mM and 0.4 mM at maximum, respectively When we assayed the activity of spinach chloroplast DHAR in the presence of 4.0 mMGSH and 0.5 mM DHA, the activity decreased by 75% upon addition of 2.0 mMGSSG, 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 mMand 0.4 mM, respectively By contrast, when we assayed the activity of chloroplast DHAR in the presence of 1.0 mM GSH and 0.1 mMDHA, the activity decreased by 40% upon addition

of 20 mMascorbate (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 mM and 2.5 mM, 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)1Æh)1in the reaction catalyzed by APX for decomposition of hydrogen peroxide at a light intensity of 1400 lmol photonsÆm)2Æs)1in 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 form ation of DHA would be 15 lmolÆmg chlorophyll)1Æh)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 mM 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 PetroleumEnergy 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 ), fromwhich 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 potassiumphosphate (pH 7.0) that

contained 1 m M EDTA under aerobic conditions For other details

see Experimental procedures.

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 fromrice.

Plant Cell Physiol 38, 173–178.

9 Hossain, M.A & Asada, K (1984) Purification of

dehydro-ascorbate reductase fromspinach and its characterization as a

thiol enzyme Plant Cell Physiol 25, 85–92.

10 Dipierro, S & Borraccino, G (1991) Dehydroascorbate reductase

frompotato 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 fromhuman 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) fromrice (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

serumalbumin 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 fromrat liver Methods

Enzymol 279, 30–35.

17 Yang, Y & Wells, W.W (1991) Identification and

characteriza-tion of the funccharacteriza-tional 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 p52 m akes it a new m em ber of

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) Metabolismof 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 fromspinach 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

Vmax

2

KDHA

i KGSH1

m ½GSH þ ðKGSH1

m þ KGSH2

m Þ½DHA½GSH þ KDHA

m ½GSH2þ ½DHA½GSH

2 ðA1Þ

and in the presence of reaction products,

m Ử

VmaxfVmaxr ơDHAơGSH2ơAsA GSSGKơ 

eq

VrKDHA

i KGSH1

m ơGSH ợ VrKGSH2

m ơDHAơGSH ợ VrKGSH1

ợVrKDHAm ơGSH2ợ VrơGSH2ợVfK

GSSG

KDHA

i Keq

ợVfK

GSSG

KDHA

i KGSH1

i Keq

ợVfK

GSSG

Keq

ợVfK

AsA

m ơGSSG

Keq

ợVfơAsAơGSSG

Keq

ợ VrKDHAm KGSH2i ơGSHơGSSG

ợVrK

DHA

i KGSH1i ơGSHơGSSG

KGSSG i

ợVrK

DHA

m KGSH2i ơGSHơAsAơGSSG

KAsA

i KGSSG i

ợVrK

DHA

m ơGSH2ơGSSG

KGSSG i

đA2ỡ

Determination of kinetic parameters

We determined Michaelis constants and kcat fromour

initial-velocity experiments using Eqn (A2) We can

trans-formEqn (A2) as follows:

vỬ

VmaxơGSH

ơGSH ợ KGSH1

m ợ KGSH2

m

 ơDHA

ơDHA ợK

DHA

i KGSH1m ợ KDHA

KGSH1

m ợ KGSH2

đA3ỡ

The plots of initial velocity at various fixed concentrations

of GSH and varying concentrations of DHA were fitted

to the MichaelisỜMenten equation

([DHA] + m2) Calculations for m1 and m2 were made

by application of the computer program KALEIDAGRAPH 3.08d, where m1 and m2 represent VmaxơGSH=đơGSHợ

KGSH1

m ợ KGSH2

m ỡ and đKDHA

i KGSH1

m ợ KDHA

đKGSH1

m ợ KGSH2

m ợ ơGSHỡ, respectively To determine

Vmax, KGSH1

m ợ KGSH2

m , KDHA

m , we generated double-reciprocal plots between m1and the concentration

of GSH, and double-reciprocal plots between m1/m2and the concentration of GSH The slope and intercept of the former plot gave Vmax and KmGSH1ợ KGSH2

m while the slope and intercept of the latter plot gave KiDHAand KmDHA

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