oxygenase

ARCHIVES OF BIOCHEMISTRY Vol. 199, No. 2, February,

AND BIOPHYSICS pp. 400-412, 1980

2-Carboxy-D-hexitol 1,6-Bisphosphate: An Inhibitor 1,SBisphosphate Carboxylase/Oxygenase’ GEOFFREY

Program

in Biochemistry

L. R. GORDON,2 V. BRYAN AND BRUCE A. MCFADDEN”

and Biophysics

and the Department Pullman, Washington Received

July

LAWLIS,

of’ Chemistry, 99164

Washington

of D-Ribulose JR.,

Sta,te University,

26, 1979

2-Carboxy-D-hexitol 1,6-bisphosphate (CHBP) has been prepared from D-fructose 1,6bisphosphate and cyanide. DEAE-Sephadex chromatography separated the reaction products into two fractions which were identified as CHBP and CHBP-lactone. CHBP is presumably a mixture of two diastereomers, 2.carboxy-D-glucitol l,6-bisphosphate and 2carboxy-D-mannitol 1,6-bisphosphate, but an attempt to separate these compounds was not successful. The material in the CHBP-lactone peak had no effect on D-ribulose 1,5-bisphosphate (RuBP) carboxylase. However, CHBP was a potent reversible inhibitor of RuBP carboxylases. This compound displayed an inhibition constant (K, at pH 8.0 and 30°C) of l-2 PM with the enzymes from spinach and barley, while the Ki was 60-70 PM with bacterial RuBP carboxylases from Pseudomonas oxalaticus and Rhodospirillum rubrum. The mode of inhibition was competitive with respect to RuBP for all the carboxylases, and noncompetitive with respect to CO, for the enzymes from spinach, P. oxalaticus and R. rubrum. The results indicate that, in the binding of certain organic phosphates by RuBP carboxylases, there may be a fundamental difference between the enzymes isolated from microbial and from higher plant sources. RuBP oxygenase activities from spinach and P. oxalaticus were also inhibited by CHBP, with K, values which were similar to those obtained with the carboxylase activity of the same enzymes. The mode of inhibition of the oxygenase activities was also competitive with respect to RuBP. Thus, it seems that the binding of CHBP is similar for the carboxylase and oxygenase reactions of the same enzyme.

D-Ribulose 1,5-bisphosphate carboxylaseioxygenase (EC 4.1.1.39) catalyzes the incorporation of a molecule of CO, into RuBP4 to yield two molecules of 3-phosphoglycerate, as well as the oxygenation of RuBP to give one molecule each of 3-phosphoglycerate and 2-phosphoglycolate. The

’ This research was supported in part by NIH Grant GM-19,972 and Hermann Frasch Foundation. 2 Present address: Department of Microbiology, Monash University Medical School, Alfred Hospital, Prahran, Victoria, Australia 3181. 3 Author to whom inquiries should be directed. * Abbreviations used: RuBP, D-ribulose 1,5bisphosphate, CRBP, 2-carboxy-D-ribitol 1,5bisphosphate; Hepes, N-2-hydroxyethylpiperazine1’-2-ethanesulfonic acid; BME, pmercaptoethanol; CHBP, 2.carboxy-D-hexitol 1,6-bisphosphate; FBP, D-fructose 1,6-bisphosphate; F6P, D-frUctOSe &phosphate. 0003-9861/80/020400-13$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

400

competing carboxylase and oxygenase reactions of this enzyme are responsible for the initial step of autotrophic CO, fixation and of

photorespiration,

respectively

(1, 2). This

branch point of photosynthetic carbon flow has been viewed as an obvious site for metabolic control in studies with CRBP (3, 4), xylitol 1,5-bisphosphate (5), and xylulose l,&bisphosphate (6), all of which appear to inhibit RuBP carboxylase and oxygenase in a time-dependent manner. Like xylulose bisphosphate, FBP can be considered an analog of RuBP, but it has only a slight effect, if any, on both enzyme activities of higher plants (4, 7,8) and on the bacterial carboxylase from Pseudomonas faciilis (2). Another potential substrate D-sedoheptulose 1,7-bisphosanalogue, phate, was recently shown to inhibit both carboxylase and oxygenase activities of a higher plant enzyme (9).

RIBULOSE

BISPHOSPHATE

CARBOXYLASEiOXYGENASE

Carboxyribitol bisphosphate is particularly interesting since it may be a transition-state analog in the carboxylase reaction (10). In view of this, the higher homolog carboxyhexitol bisphosphate has been prepared from FBP and cyanide. We now describe the synthesis, isolation of the products, ant1 their effects upon RuBP carboxylaseioxygenase from some plant and bacterial sources. MATERIALS

AND

METHODS

Materials D-Ribulose 1,5-bisphosphate (tetrasodium), Dfructose 1,6-bisphosphate (tetrasodium), D-fructose 6phosphate (disodium), disodium %phosphoglycerate, D-fructose, spinach RuBP carboxylase, crystalline bovine serum albumin, rabbit muscle FBP aldolase, glycerol :%phosphate dehydrogenaseitriose phosphate isomerase, NADH (tlisodium), and Hepes were all purchased from Sigma. Ultrapure ammonium sulfate and Tris. as well as NaH’“CO,, (53 Ci/mol), xvrre products of SlvarziMann. ICN supplied Na”CN (3 Ci,‘mol). while K’:‘CN (90 atom%) came from Prothem. DEAE-Sephades A-25 and Sephadex G-25 (fine) \vere obtained from Pharmacia, while Amberlitr lR120 (hydrogen form) was obtained from Fisher. Deuterium oxide and 8-valerolactone were purchased from Aldrich. %Carbosyhexitol (fructoheptonic acid) was synthesized via the cyanohydrin of D-fructose (ll), except that the decolorization and crystallization steps \vere omitted. The reaction mixture was bound to a DEAE-Sephades A-23 column and other components removed by exhaustive washing \vith 10 mM NH,HCO,, until the effluent was clear. The product of interest nx~ elutrd by 4-5 column vol of 100 mM NH,HCO,,, subjected to rotary evaporation, and the syrup treated alternately with methanol and acetone followed in each case by rotary evaporation. This m-as repeated until the odor of ammonia could not be detected. The product xas lyophilized to yield a thick, syrupy bro\\n substance \vhich \vas converted to the Lisalt on an Amberlite IR-120 column in the Li-form. Finally, the product was decolorized with Norite A and lyophilized. All other chemicals \vere of the highest grade available. Solutions Lvere prepared Lvith glass distilled xvater.

Synthesis nlld Purification of &Carbory-u-hexitol 1,6-Bisphosphate CHBP was prepared from FBP by means of the qwwhydrin reaction and hydrolysis of the resulting nitrile by a modification of the method used fol

INHIBITOR

401

the preparation of CRBP (10). FBP (1 mmol) was incubated with 1.5 mmol KCN in 10 ml water contained in a Thunberg tube at 23°C for about 16 h. Carboxy[14C]CHBP (and the related lactone) was prepared by adding Na“‘CN (10 FCi) to the reaction mixture. while carboxy-[‘:‘C]CHBP (and its la&one) was prepared with Kl:‘CN (90 atom%). The pH of the mixture was about 9.5 after reaction and was adjusted to approximately 7.5 \vith HCI with subsequent magnetic stirring at 23°C for 1 h to reduce the amount of cyanide present. If a crude CHBP preparation was desired, the reaction mixture xcas applied to a lo-ml Amberlite IR-120 (lithium form) column and eluted with distilled \vater. The crude mixture of lithium salts of CHBP and the la&ones was isolated by lyophilization. The solid material \vas washed extensively with methanol: acetone (1%. v/v) to remove excess LiCl and stored in C~CCILO over P,O, at 23°C. Alternatively purification of the products \vas achieved by applying the reaction mixture to a DEAE-Sephades A-25 column (2.5 x 40 cm) which had previous1.v been equilibrated with 0.1 M NH,HCO,, at 4°C. The column was then eluted with a l-liter linear gradient from 0.1 to 0.5 M SH,HCO:,. Desired fractions \vere pooled and separately evaporated to dryness at 30°C under reduced pressure. The residues were dissolved in about 10 ml distilled water, filtered through Whatman No. 1. and then converted to the lithium salts. lyophilized, xvashed. and stored as described earlier. Separation of the putative diastrreomers of CHBP leas attempted hy chromato~raphg of this material at 4°C on a Dowex-1 (formate form) column (0.9 X 22 cm) followd by elution \\ith a linear grxlient consisting of 1 liter of distilled water and 1 liter of 0.4 x formic acid and 0.75 N sodium formate at a flo\v rate of 60 ml/h.

‘T-NMR

Measurements

l:‘(‘-NMR spectra were obtained at 22.62 MHz on a Bruker WH 90 spectrometer which was equipped with a B-NC 12 computer with a maximum 20,000 data memory bank. The lo-mm standard Bruker probe was used. Broad-band decoupling for protons \vas used to collect spectra using ‘i kHz sweep width with 5000 to 22.000 s\z-eeps and 8000 data points per spectrum at 27°C. Except as indicated, the natural abundance of ‘:‘C was measured in compounds analyzed. The standard used was 3(trimethylsilyl)propionic acid.

Analytical

Methods

D-Fructose 1.6.hisphosphate \vas determined enzymatically using FBP aldolase, triosephosphate isomerase. and glycerol Z-phosphate dehydrogenase (12). Ammonia \vas determined calorimetrically \vith Nessler’s reagent (13). The inorganic and total phosphate content of phosphorylated compounds was determined by the method of ilmes (14).

402

GORDON,

LAWLIS,

Carboxylic acid ester (and lactone) functional groups were determined calorimetrically using a modification of the procedure described by Peel (15). Because of the phosphorylated nature of the compounds tested, the following procedure was found to be necessary. Hydroxylamine and NaOH solutions were added in that order to the sample solution containing the ester bond, which was then allowed to stand at 23°C for exactly 10 min prior to acidification with HCl and addition of FeCl, solution. It was imperative to measure the absorbance at 505 nm immediately, before a precipitate formed. Freshly prepared aqueous solutions of &valerolactone were used as standards, since it was noticed that older solutions (even when they had been stored at -20°C) did not produce as much color in the assay. Cyanide was determined calorimetrically using the cyanogen bromide reaction and benzidine dihydrochloride (16). Protein concentrations were determined colorimetrically by a modification (17) of the Folin method (18). Crystalline bovine serum albumin was the standard.

Sources

of RuBP

Carborylases

Rhodospirillum rubrum (a photosynthetic, purple nonsulfur bacterium) was grown on butyrate-minerals in the light as previously described (19), harvested by centrifugation, and stored at -20°C until used. One gram of thawed cells was suspended in 10 ml buffer A (50 mM Tris-Cl, pH 8.0, at 23”C, 1 mM disodium EDTA, 10 mivr MgCl,, 50 mM NaHCO,, and 5 mM BME), and sonically treated for six 30-s periods at 0°C. The supernatant solution, after centrifugation at 160,OOOg for an hour, was fractionated between 30 and 60% saturated ammonium sulfate at 0°C. Prior to use for RuBP carboxylase assays, the preparation was passed through a small Sephadex G-25 column (0.7 x 10 cm) which had previously been equilibrated with buffer A. The specific activity of the crude enzyme preparation was 0.1 pmol CO, fixedlminimg. RuBP carboxylase was purified to homogeneity from formate-grown Pseudomonas oxa2aticus as previously described (20). The enzyme was stored in buffer B (50 mM Hepes, pH 8.0, at 23”C, 1 mM EDTA, 10 mM MgCl,, 50 mM NaHCO,, and 5 mM BME) and had a specific activity of 1.5 pmol CO, fixediminimg protein when assayed immediately after purification. Barley (Hordeurn vulgnre) RuBP carboxylase was generously supplied by A. K. Saluja of this laboratory and had been purified as previously described (9). The preparation used in this study had a specific activity of 0.5 Fmol CO, fixediminimg. A commercial preparation of partially purified RuBP carboxylase from spinach (Spinacia olerucea) was dissolved in buffer A and further purified by centrifugation in linear sucrose density gradients (21). Sucrose was removed from the pooled fractions by adsorption

AND

MCFADDEN

of the enzyme onto a small DEAE-cellulose column (0.7 x 6 cm) followed by elution with buffer A containing 0.15 M NaCl. The final specific activity was 1.6 pmol CO, fixedlminimg, after removal of NaCl from the preparation by passage through a small Sephadex G-25 column which had been equilibrated with buffer A.

Assay

of RuBP Carboxylase Activities

and

Oxygenase

In early experiments, RuBP carboxylase activity was measured in a mixture containing: 64 mM TriCl (pH 8.0, 23°C). 8 mM MgCl,, 20 mM NaH14C0, (0.3 Ciimol). Enzyme preparations were incubated in 200 ~1 of this mixture for at least 5 min at 30°C prior to initiation of the assay with 0.8 mM RuBP to give a final volume of 250 ~1 (22). In later kinetic experiments for the determination of inhibition constants, enzyme preparations were equilibrated with buffer C, pH 8.0, at 23°C (50 mM Tris-Cl, 1 mM EDTA, 10 mM MgCl,, 20 mM NaHCO,) by passage through a small Sephadex G-25 column, and incubated for at least 5 min at 30°C prior to initiation of the assay which contained the same concentration of all components, except where noted. After 1 min at 3O”C, the assays were terminated with 100 ~1 of 60% cold trichloroacetic acid. Unreacted “‘CO, was liberated from a 200-+l sample by heating at 70-85’C for 45 min followed by addition of scintillation cocktail (23) and counting. RuBP oxygenase activity was determined with a Hansatech (England) oxygen electrode in a 0.5.ml vessel, taking the precautions suggested previously (24). The apparatus was fitted with a custom-made amplifier which expanded 92-115 nmol0, to full scale. Enzyme was equilibrated with buffer C and incubated at 30°C for at least 10 min before addition of a 25~1 aliquot to the oxygenase assay system. The activated enzyme preparation was used to initiate the reaction at 30°C in which assay mixtures contained 50 mM Tris-Cl (pH 8.0 at 23”C), which had been saturated by CO,free air as well as 8 mM MgCl, and RuBP concentrations as indicated. The concentration of CO, in the assay solutions had been reduced by preparing them with water which had been acidified, boiled, and cooled. The rate of oxygen consumption was linear with time for about the first minute, and was always dependent upon addition of RuBP.

Determination

of Enzyme Inhibition

Constants Inhibition constants (K,) for competitive and noncompetitive inhibition were determined graphically from Dixon plots (25, 26). Lines of best fit were determined by linear least squares analysis, USing data which had been generated with two substrate concentrations.

RIBULOSE

BISPHOSPHATE

CARBOXYLASEiOXYGENASE:

RESULTS

Characterization of 2-Carboxy-D-hexitol 1,6-Bisphosphate

Enzymatic analysis of the commercial FBP used for the preparation of CHBP revealed a purity of 90%. Using this value to calculate the concentration of FBP in the reaction mixture, coupled with the amount of ammonia liberated in the reaction of FBP with cyanide by the hydrolysis of the resulting cyanohydrin, the stoichiometry of the reaction could be calculated and fell within the range of 0.90 to 0.95. Due to the lack of chemical specificity of the reaction, it was presumed that the product would be a diastereomeric mixture of 2-carboxy-& glucitol 1,6-bisphosphate and 2carboxy-Dmannitol 1,6-bisphosphate. Hence, the generalized name 2-carboxy-D-hexitol 1,6bisphosphate (CHBP) was used to account for this possibility. CH,OPO:C(OH)CO,

I

HO- C-H

I

H-C-OH I H-C-OH I CH,OPO;2-Carboxy-D-hexitol

1,6-Bisphosphate

Recovery of the penta lithium salts of crude CHBP was nearly theoretical (based on a molecular weight of 416). Further evidence for the successful reaction of FBP with K14CN was obtained when there was no loss of radioactivity after treatment of the products with concentrated HCl. In addition, the products of the reaction failed to produce color with reagents such as Z,bdinitrophenylhydrzine, orcinol, and carbazole, indicating the loss of carbonyl function and modification of the carbon-2 of FBP. Determination of organic phosphate in the crude preparation revealed slightly less than two per CHBP molecule, based on a molecular weight of 416. The isolated crude prepara-

INHIBITOR

403

tion of lithium CHBP contained only traces of FBP and cyanide. Purification of reaction products was conducted on DEAE-Sephadex columns using gradient elution with NH,HCO,,. Typical elution profiles are shown in Fig. 1. Three phosphate-containing peaks were evident, which coincided precisely with the three radioactive peaks when carboxy-labeled products had been chromatographecl (Fig. 1A). These peaks were termed I, II, and III in their order of elution from the column at approximately 0.2, 0.25, and 0.3 M NH,HC03, respectively. In a duplicate experiment (Fig. lB), fractions were assayed for esters and a peak coinciding with II was found. When FBP was chromatographed under identical conditions (Fig. lB), it eluted at nearly the same point as peak II, whereas fructose 6-phosphate eluted before peak I. A number of conclusions significant to the remainder of the paper were drawn from these results. Peak III was more retarded on the column than FBP, indicating additional negative charge in it, vvhile peak II had a similar charge to FBP and comprised ester(s). Therefore, it seemed likely that peak III was CHBP and that peak II was a lactone of CHBP involving the introduced carboxyl group at carbon-2 and, presumably, the hyclroxyl at carbon-5 to form the six-membered cyclic structure shown below:. CH,OPOi?,I C(OH)CO I HO- C-H I H-C -OH I H-C--O

I

CH,OPO;2Carboxy-D-hexitol

1,6-BisphosphateS-la&one

Based on the calorimetric estimation of esters and total phosphate, the peak fraction of II (Fig. 1B) contained 6.2 mM phos-

404

GORDON,

LAWLIS,

1,200 11,000 1800 600 1

AND

MCFADDEN

F6P

FBP

1400

200 1 0

Elution

Volume

(liters)

Elution

Volume

( liters

1

FIG. 1. Chromatography of reaction products on DEAE-Sephadex. The columns (25 x 40 cm) were developed with Q-liter linear gradients from 0.1 to 0.5 M NH,HCO, at 4°C; 25-ml fractions were collected at a flow rate of 180 ml/h. Carboxylic acid esters and total phosphate were determined colorimetrically as described at 505 and 800 nm, respectively. (A) Separation of products obtained from condensation of 0.5 mmol of FBP and 0.75 mmol of K’“CN (1.4 mCi/mol). (B) Separation of 1.5 mmol CHBP. The bars indicated were fructose 6-phosphate and FBP eluted under the same conditions of separation.

phate and 1.2 mM ester which gave a phosphate to ester ratio of 5-2, much above the ratio of 2 expected on a theoretical basis. This lack of a stoichiometric relationship may have been partially due to different light absorption properties of the hydroxamate-Fe& complex of the &valerolactone standard used in the calorimetric ester determination. It was also possible that after chromatographic separation of CHBP-lactone, some reverted to the original CHBP. In either case, the test could only be regarded as semiquantitative. Traces of FBP were found in CHBPlactone preparations which could be expected as the chromatographic procedure would not separate them. Peak I, containing both organic phosphate and 14C label derived from K14CN and eluting slightly later than fructose 6-phosphate, was possibly the carboxylated derivative of the fructose &phosphate which was present in low quantities as a contaminant of the commercial FBP. Confirmation of the identity of the phosphate-containing peaks eluted from DEAESephadex columns was undertaken by the use of natural-abundance 13C-NMR spectroscopy. Indeed, isolated CHBP gave two signals (180.9 and 180.4 ppm) which were in the region where resonance due to a carboxylic acid group would be expected (Fig.

2A), in consonance with the formation of diastereomeric products. When acidified (3 eq H+/mol of CHBP) under conditions in which the lactone(s) could form, the two signals shifted to 177.5 and 177.2 ppm and the lactone resonance was seen at 179.2 ppm. Indeed the lactone could be detected colorimetrically. A shift upfield accompanying conversion of carboxylates to carboxylic groups is expected (27) and was confirmed in studies of 3-phosphoglycerate and the parent carboxylic acid in which a shift from 180.5 to 177.2 ppm was observed. The two doublets seen at 84.4 and 80.4 ppm (Fig. 2A) were presumably due to the Cr carbon of each diastereomer with each doublet arising from splitting of the ‘“C resonance by the 31Pnucleus on the adjacent carbon (28). Indeed resonance in this region was absent from 3-phosphoglycerate. Two singlet signals were, moreover, present in the 2-carboxyhexitol sample at 85.3 and 82.0 ppm as was a carboxglate peak at 180.8 and a small signal slightly downfield reflecting the expected diastereomeric composition of this substance (Fig. 2C). When chromatographic peak 2 \vas examined 6 months after isolation, a small lactone resonance at 180.2 ppm was observed with the two carboxylate resonances at 180.9 and 180.5 ppm. Again two doublets were observed at 84.0 and 80.8 ppm (Fig.

RIBULOSE

BISPHOSPHATE

CARBOXYLASEiOXYGENASE

INHIBITOR

FIG. 2. Natural-abundance ‘“C-NMR spectra recorded at 22.62 mHz. Aqueous solutions (il n 99% 2H,0) were: (A) peak III (0.63 M), 11 months after isolation; (B) peak II (0.78 M), 6 months after isolation; (C) lithium carboxyhexitol (a saturated solution), 15 months after isolation. Abscissa units for chemical shifts are in parts per million (ppm).

405

406

GORDON, LAWLIS, AND MCFADDEN

ZB). The area under the latter doublet was larger and presumably reflected coincidence of the signals for C, of the la&one with those of one of the diastereomeric salts. When peak II was acidified with 3 eq H+/ mol of CHBP-lactone, again the pronounced chemical shift upfield of two resonances was noted to 178.4 and 177.4 ppm with little alteration in the lactone resonance which was 179.5 ppm. Thus, it seemed that after elution of CHBP-lactone from the DEAESephadex column some spontaneous hydrolysis of the ester bond had occurred producing a mixture of CHBP and CHBPlactone before the phosphate-containing peak II could be isolated. This possibility was confirmed when peak II material from DEAE-Sephadex chromatography (Fig. 1B) was concentrated by rotary evaporation under reduced pressure immediately after elution from the column, converted to the lithium salt, lyophilized, and then rechromatographed on DEAE-Sephadex. Two phosphate-containing peaks were found which corresponded precisely to those in Fig. 1B. This finding, at least in part, explained the lack of stoichiometry found earlier between phosphate and ester in peak II material. Samples of CHBP applied to a Dowex-1 (formate-form) column in an attempt to separate the putative diastereomers, were eluted as single broad peaks at approximately 0.2 N formic acid and 0.5 N sodium formate. Nature of the Inhibition of RuBP Carboxylase by CHBP

Preliminary experiments on the effect of the crude reaction products (as the lithium salts) on RuBP carboxylase were performed with the purified enzyme from P. oxalaticus. When the enzyme was incubated in the presence of magnesium and bicarbonate (Buffer A) at 30°C with these products at 1 mM and then assayed at various time intervals over a total period of an hour, the degree of inhibition was independent of the incubation time. The observed inhibition could not be attributed to contaminating cyanide or FBP. A second test involved

incubation of the P. oxalaticus enzyme in the presence of magnesium and bicarbonate (Buffer A) with and without 1 mM crude reaction products at 4°C. After 24 h, the two samples were gel-filtered and assayed. The specific activities of the enzyme after the different treatments were identical. Thus, it seemed that the mixture of CHBP and CHBP-lactone is a reversible inhibitor of RuBP carboxylase from P. oxalaticus. The three fractions obtained upon chromatography on DEAE-Sephadex (see Fig. 1) were tested as inhibitors of RuBP carat 0.3 mM boxylase from P. oxalaticus concentration in the assay. Peak I was not inhibitory, while peaks II and III inhibited 27 and 46%, respectively. However, peak II had already been shown to consist of a mixture of CHBP and CHBP-la&one, so the inhibition may not have been due to CHBP-lactone. When samples of the peak fraction from both II and III (Fig. 1B) were separately added to assays of RuBP carboxylase from P. oxalaticus the data summarized in Table I were obtained. Peak II material inhibited enzyme activity, and this inhibition increased over a 2-day period when the material was stored at 4°C in the concentration of NH,HCO, which caused its elution (approximately 0.22 M). However, when the NH,HCO, was removed from a sample of peak II material by rotary evaporation under reduced pressure, it inhibited RuBP carboxylase to the same degree as the untreated sample, indicating that this evaporation step in the recovery process was not responsible for the change in the proportions of CHBP and CHBP-lactone in the mixture. The inhibitory effect of peak III material (CHBP) remained unchanged with storage time. Table I also shows the inhibition of RuBP carboxylase caused by the concentrations of CHBP that might have been added with the peak II material (footnote c). This inhibition may account for the inhibition observed with peak II material. More definitive information on the inhibitory effect of CHBP-lactone was obtained when carboxy-[13C]CHBP (and its lactone) were synthesized, chromato-

RIBULOSE BISPHOSPHATE

CARBOXYI,ASE/OXYGENASB

407

INHIBITOR

T.4BLE I INHIBITION OF RIBULOSE BISPHOSPHATECARBOXYLASEFROMPseudomonas oxalaticus BY PRODUCTS OF THE REACTIONOF FRUCTOSEBISPHOSPHATEWITH CYANIDE AFTER THEIR ELUTION FROM.4 DEAE-SEPHADEX COLUMN

Peak II in NH,HCO,, solution II with NH,HCO:, removed” III in NH,HCO,, solution

Percentage inhibitionb Time at 4°C after separation

Concentration m assay” (mm

Same day

1 Day

0.13 0.30

10’ 21

18

22

27

32

ND’ ND

ND ND

0.13

8

2 Days

0.26

“”

0.15 0.80

BY

23

29

48

47

4:3

I’ Percentage inhibition relative to control assays conducted without inhibitor. The enzyme specific activit) in these controls K~S 1.5 wmol CO, fixediminimg. Enzyme activities in the presence of inhibitor lvere corrected for bicarbonate added \vith the inhibitor solution. b The concentration was determined from total phosphate analysis assuming two phosphates per molecule. ’ Peak II contained 1.5 mM bisphosphorylated compounds (see footnote b) and 0.5 mM ester (as CHBPlactone). so by difference it may have also contained 1.0 mM CHBP. Therefore it was necessary to test the inhibition caused by 0.1 and 0.2 mM CHBP (isolated as peak III). This inhibition was 9 and SO%,respectively. ” NH,HCO,, was removed bv rotary evaporation at 30°C under reduced pressure for 2 h. 1’Not determined.

graphed on a DEAE-Sephadex column, and eluted with a 4-liter linear gradient from 0 to 0.73 M NH,Cl (which had been adjusted

to pH 6.5 with NH,OH). Three peaks were again observed, with peaks II and III being eluted at approximately 0.2 and 0.23 M

(A)

-02

[CHBP]

mM

[CHBP]

mM

FIG. 3. Inhibition of RuBP carbosglase from P. ornlaticus by CHBP with RuBP as the variable substrate. (A) Activation of the enzyme in the assay followed by initiation with RuBP. The enzyme (2.5 @,I$\vhich had been stored for .5days after preparation was incubated in the assay mixture plus 01 minus CHBP containing 0.X mM CO2for 5 min prior to addition of RuBP. (B) Initiation with activated enzyme. The assays, which contained a total of 0.7 mM CO, and RuBP as indicated, were initiated by the addition of 21 p,g of preactivated enzyme so that CHBP (urhen present) was added at time zero. This enzyme batch had been stored for 6 days after preparation.

408

GORDON,

LAWLIS,

AND MCFADDEN

NH,Cl, respectively. Samples of each peak were tested as inhibitors of RuBP carboxylase from P. oxalaticus in assays containing up to 2.0 and 1.0 mM bisphosphorylated compound for peaks II and III, respectively. Peak III (CHBP) inhibited 62% at 1.0 mM, while peak II (CHBP-la&one) was not inhibitory at a concentration of 2.0 mM. In addition a sample of the carboxy-[‘“ClCHBP-la&one (peak II) was analyzed with the ‘“C-NMR spectrometer. In the la&one region a large signal with a small upfield shoulder was observed, presumably due to formation in minor amounts of the diastereomeric la&one, which was not detected in the natural abundance ‘“C spectra. Because CHBP-la&one(s) were not inhibitory, only CHBP was subjected to further study.

-4

0

4

8

[cHBP]~M

-40

-30

-2o=

0

IO

20

30

[CHBP]~M

FIG. 4. Inhibition of spinach RuBP carboxylase by CHBP with RuBP (A) or CO, (B) as the variable substrate. All assays were initiated by 24 kg of preactivated enzyme to RuBP (plus CHBP when present) and, in addition to the indicated substrates, contained a total of 0.7 lllM CO, (A) or 1.0 mM RuBP (B).

Inhibition of RuBP Carboxylasel Oxygemse by CHBP

The inhibitory effect of CHBP was investigated with RuBP carboxylase from R. mbrum, P. oxalaticus, barley, and spinach. Representative data for the inhibition with RuBP as the variable substrate are shown as Dixon plots in Figs. 3A, 3B, and 4A. The data displayed in Fig. 3A were generated by activating the enzyme from P. oxnlaticus followed by simultaneous ad-

dition of CHBP and RuBP (see Materials and Methods). Although plots were biphasic, essentially the same K, value for CHBP was obtained (see lower limbs) when the assays were initiated with preactivated

TABLE

II

SUMMARY OF INHIBITION CONSTANTS FOR ~XARBOXYHEXITOL 1,6-BIsPHOSPHATE WITH RIBULOSE BISPHOSPHATE CARB~~~LA~E~O~~GENA~E K&M

CHBP)”

Variable

RuBP

Carboxylase Source of Enzyme Rhodospirillum rubrum Pseudomonas oxalaticus Barley Spinach

Variable 340 270 ND 40

CO,

60 65 2 1.0

Oxygenase Variable RuBP ND” 55 ND 1.2

(’ All K, values were determined graphically from Dixon plots using either two concentrations of RuBP or CO,. CHBP was a competitive inhibitor with respect to RuBP for both carboxylase and oxygenase activities, and noncompetitive with respect to CO, for the carboxylase activity. b Not determined.

RIBULOSE

BISPHOSPHATE

CARBOXYLASEiOXYGENASE

INHIBITOR

409

the Dixon plot with variable RuBP biphasic and in both eases the inhibitor was competitive with respect to RuBP. These results are summarized and compared to those obtained for RuBP carboxylases in Table II. DISCUSSION

-01

0

02

01 [CHBP]

mM

FIG. 5. Inhibition of RuBP oxygenase from P. oralaticus by CHBP with RuBP as the variable substrate. Each assay contained RuBP and CHBP (when present) as well as 30 pM CO, which had been carried over \vhen the assay was initiated with 20 kg of the preactivated enzyme (specific activity = 1.5 pmol CO, fisediminlmg). This enzyme preparation had been stored for 7 days after purification. The initial concentration of Or was 262 pM.

enzyme (Fig. 3B). In contrast, plots reflecting analogous assays of the spinach enzyme (Fig. 4A) were not biphasic. This general approach allowed unambiguous comparison of the Ki values obtained for the carboxylase and oxygenase reactions. Data for the barley enzyme (not shown) were generated similarly to those foi spinach RuBP carboxylase, while data for the R. rubrum enzyme (not shown) were obtained at a constant CO, concentration of 1.0 m&t with 0.05 and 0.15 rnM RuBP. In all cases, the mode of inhibition by CHBP was competitive with respect to RuBP. A summary of the Ki values obtained from Dixon plots when RuBP was the variable substrate is given in Table II. When CO, (provided as NaHCO,) was the variable substrate, CHBP inhibited spinach RuBP carboxylase noncompetitively with respect to CO, (Fig. 4B). In addition, Dixon plots revealed that CHBP was a noncompetitive inhibitor with respect to CO, for RuBP carboxylases from R. rubrum and P. oxalaticus (data not shown). RuBP oxygenase from P. oxalaticus or spinach was also inhibited by CHBP (see Fig. 5 and Table II). In neither case was

CHBP and CHBP-lactone, the products of the reaction of FBP with cyanide, could be separated by the basis of charge by DEAE-Sephadex chromatography under slightly alkaline conditions. However, by the time these separated products could be isolatecl, the CHBP-la&one fraction was found to be a mixture of CHBP-lactone and CHBP, while the composition of the CHBP fraction remained unchanged during the isolation procedure. In the analogous preparation and purification of CRBP by the reaction of RuBP with cyanide, DEAEcellulose chromatography separated the reaction products into a major peak which had a spur on the leading edge (10). The position and magnitude of the spur would be consistent with it being the y-lactone of CRBP. Subsequent experiments with the material isolated from the main peak revealed that 91 to 93% of the CRBP added to spinach RuBP carboxylase over a wide range of subsaturating concentrations bound to the enzyme and a constant percentage (7 to 9%) remained free (3). This finding was explained as the result of a stereochemical preference in the cyanohyclrin reaction with selective binding of the predominant diastereoisomer of CRBP to spinach RuBP carboxylase (10). On the basis of our findings that CHBP-lactonecontaining material could be separated from CHBP by conventional ion-exchange chromatography and that CHBP-lactone is noninhibitory toward RuBP carboxylase from P. oxalaticus, it seems that the same may have been true for CRBP. CHBP was an extremely potent inhibitor of RuBP carboxylase. There were some characteristics of this inhibition Lvhich served to distinguish it from the inhibition caused by other potent inhibitors of RuBP carboxylase. Unlike CRBP (3, 4), xylitol bisphosphate (5), ancl xylulose bisphosphate

410

GORDON.

LAWLIS,

(6), CHBP inhibited plant and bacterial enzymes in a manner which was independent of the contact time between the inhibitor and the enzyme. Although a mixture of CHBP and CHBP-lactone slightly inhibited activation of the enzyme from P. oxalaticus by magnesium ion and CO, (unpublished observation; see Ref. (29) for general assay conditions), the inhibition was not nearly sufficient to account for the inhibition observed in the early experiments. This possibility has been recognized for some other inhibitors of RuBP carboxylaseloxygenase, but, in general, effects upon activation have not been excluded. The high Ki values of CHBP observed in Dixon plots with variable CO, concentrations as compared to those values obtained in a similar manner with high CO, and variable RuBP concentrations (Table II), suggest an interesting problem. Perhaps there is potentiation of activation of the carboxylase by CHBP in the presence of low CO, concentrations, similar to that observed for 6phosphogluconate with RuBP carbosylase from P. oxalnticus (29), which would be manifested in an increased Ki for CHBP (cf. Figs. 4A and B). The change in Kj values for CHBP with variable CO, for the enzymes from P. oxalaticus and R. rubrum are small when compared with the change for spinach RuBP carboxylase. Therefore it seems that there may be a substantial activation of spinach enzyme by CHBP at low CO, which merits further study. RuBP oxygenase is primarily responsible for photorespiration, a process which is detrimental to net photosynthetic CO, assimilation and consequently to plant productivity (for reviews see (30, 31)). Various workers have sought compounds which would selectively inhibit the oxygenase activity of RuBP carboxylaseioxygenase (4, 32, 33), in order to gain further knowledge of the two reactions. Along these lines, CHBP was originally synthesized as a homolog of the putative transition stateanalog CRBP of the carboxylation of RuBP. Therefore, a possibility existed that CHBP would inhibit only the RuBP carboxylase reaction (or more probably inhibit the

AND MCFADDEK

carboxylase more than the oxygenase), since the oxygenase reaction could involve a different kinetic mechanism from that of the carboxylase reaction. However, in the case of the enzymes from P. oxalaticus and spinach, CHBP inhibited both carboxylase and oxygenase activities similarly as indicated by the Ki values obtained when RuBP was the variable substrate (see Table II). In addition, CHBP was a competitive inhibitor with respect to RuBP for both carboxylase and oxygenase activities, while its inhibition was noncompetitive with respect to CO, for the carboxylase reaction. Thus, it seems that CHBP binds at the RuBP site and exerts its inhibitory effect as a substrate analog. Both xylitol bisphosphate and xylulose bisphosphate (5, 6) generated biphasic Dixon plots after incubation with spinach RuBP carboxylase prior to assay, which suggested two types of inhibition. Hovvever, the inhibitions were time dependent. Use of Dixon plots in kinetic analysis of a ligand assumes that ligand binding is rapid and reversible. The time dependence observed implies that the interaction of these ligands with the spinach enzyme was neither rapid nor reversible, suggesting that the biphasic Dixon plots Lvere artifacts of time-dependent inactivation. In our study, biphasic Dixon plots with the reversible inhibitor CHBP were found only for the enzyme from P. oralaticus and then only when the activated enzyme was used to initiate the assay, rather than initiating the reaction of activated enzyme by the simultaneous addition of CHBP and RuBP (compare Figs. 3A and B). It is also interesting to note that Dixon plots reflecting the inhibition of RuBP carboxylase from P. oxalaticus by 6phosphogluconate were biphasic when the assays were initiated with activated enzyme (Lawlis, Gordon, and McFadden, unpublished). The data then suggest that the biphasic character of CO, fixation catalyzed by the P. oxalaticus enzyme depends in some manner upon the conditions of incubation of these ligands with the enzyme. A single two-state model is presented below for CHBP.

RIBULOSE

BISPHOSPHATE

I E,,,

CARBOXYLASEiOXYGENASE

INHIBITOR

411

II

(5 min) CHBP 2 \

E,,. - CHBP

2 PGA (CHBP competes with RuBP, Ki)

(CHBP competes with RuBP, K:) K:

According to this model, 5-min preincubation \vith CHBP converts most or all activated enzyme E.,. (I) to state II. When, however, CHBP is added at zero time in the assay (Fig. 3B), only a portion of state I is converted to II during the 1-min assay resulting in the biphasic dixon plots. It is most plausible that in the formation of state II, CHBP binds at a site distinct from the catalytic site. Repeated experiments with the P. ornlaticus enzvme have established that the monophaiic and biphasic responses (cf. Figs. 3A, 3B, and 5) to CHBP are not related to the postpurification age of the enzyme, a parameter known to affect activation by CO, and Mgt2 (20). In contrast to RuBP carboxylase from P. oxalaticus, studies with variable CHBP added at zero time in assays of the spinach, barley, and R. rubrum enzymes reveal monophasic Dixon plots. Thus these other enzymes may either lack a separate CHBP binding site or the conversion of state I to state II may be much faster than for the pseudomonad enzyme. It is of interest that a monophasic Dixon plot was obtained when variable CHBP was added at zero time in assays of the second activity of the homogeneous pseudomonad enzyme, RuBP oxygenase. The oxygenase, however, was assayed in the presence of 30 PM CO, and 262 PM 0, at 23°C in contrast to 700-800 FM CO, and 230 PM 0, used in the carboxylase assays (at 30°C). Although fully activated enzyme was employed in both assays, 0, was limiting in the oxygenase measurements whereas CO, was saturating with respect to carboxylase. Thus detailed comparisons in terms of the foregoing model should be avoided.

> Ki

In an evolutionary context, RuBP carboxylases are thought to have been highly conserved (2, 34, 35). The large subunits (approximate M, 55,000), which contain the catalytic and most probably the activation sites, have so far been found in all eucaryotic and procaryotic RuBP carboxylases. K,,, (RuBP) values for properly activated spinach and barley RuBP carboxylases and tobacco RuBP oxygenase were 20 (l), 100 (9) and 22 to 28 WM (1, 36), respectively, while RuBP carboxylase from R. rubrum had a value of 53 PM (37). The RuBP carappears to boxylase from P. oralaticus have an unusually high K,,, of about 220 PM RuBP (Lawlis, Gordon, and McFadden, unpublished). When the ratio of K,,, (RuBP) to Ki (CHBP) was determined for the respective RuBP carbox?lases using unpublished data and the data m Table II, the following were obtained: spinach, 20; barley, 50; P. oxalaticus, 3; R. rubrum, 1. Thus, CHBPwas a far more potent inhibitor of higher plant RuBP carboxylases than of the bacterial enzymes, as was initially indicated by the different Ki values obtained. Since CHBP appeared to act as an analog of RuBP, the differences in inhibition may reflect major differences between plant and bacterial RuBP carboxylases with respect to the binding of RuBP. ACKNOWLEDGMENTS spectrometer was purchased The ‘:‘C-NMR with funds awarded jointly to the Chemistry Departments of Washington State University and the University of Idaho by an NSF departmental instrument grant. We wish to thank Don Appel for obtaining the spectra, and Dr. Jim Magnuson and Ed Pandolfino for assistance with interpretation.

412

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LAWLIS,

AND MCFADDEN

REFERENCES JENSEN, R. G., AND BAHR, J. T. (1977) Annu. Rev. Plant Physiol. 28, 379-400. MCFADDEN. B. A. (1973) Bacterial. Rev. 37, 289-319. SIEGEL, M. I., AND LANE, M. D. (1972) Biochem. Biophys. Res. Commun. 48, 508-516. RYAN, F. J., AND TOLBERT, N. E. (1975) J. Biol. &em. 250, 4234-4238. 3. RYAN, F. J., BARKER, R., AND TOLBERT, N. E. (1975) Biochem. Biophys. Res. Commun. 65, 39-46. 6. MCCURRY, S. D., AND TOLBERT, N. E. (1977) J. Biol. Chem. 252, 8344-8346. 7. BUCHANAN, B. B., AND SCHURMANN, P. (1973) J. Biol. Chem. 248, 4956-4964. 8. CHU, D. K., AND BASSHAM, J. A. (1974) Plant Physiol. 54, 556-559. 9. SALUJA, A. K., AND MCFADDEN, B. A. (1978) FEBS Lett. 96, 361-363. 10. SIEGEL, M. I., AND LANE, M. D. (1973) J. Biol. Chem. 248, 5486-5498. 11. SCHMIDT, 0. T., AND WEBER-MOLSTER, C. C. (1935) Justus Liebigs Ann. Chem. 515, 43-64. 12. MICHAL, G., AND BEUTLER, H.-O. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 3, pp. 1314-1319, Academic Press, New York. 13. SKERMAN, V. B. D. (1967) A Guide to the Identification of the Genera of Bacteria, p. 274, Williams & Wilkins, Baltimore. 14. AMES, B. N. (1966) in Methods in Enzymology (Neufeld, E. F., and Ginsberg, V., eds.), Vol. 8, pp. 115-118, Academic Press, New York. 15. PEEL, J. L. (1951) Biochem. J. 49, 62-67. 16. ALDRIDGE, W. N. (1944) Analyst 69, 262-265. 17. KENNEDY, S. I. T., AND FEWSON, C. A. (1968) Biochem. J. 107, 497-506. 18. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,

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