JOURNAL OF BACTERIOLOGY, May 1972, p. 633-642Copyright 0 1972 American Society for Microbiology

Vol. 110, No. 2Printed in USA.

Ribulose Diphosphate Carboxylase fromAutotrophic Microorganisms

BRUCE A. McFADDEN AND ANITA R. DENENDI

Department of Chemistry, Washington State University, Pullman, Washington 99163

Received for publication 20 January 1972

Thiobacillus denitrificans was grown anaerobically with nitrate as an ac-

ceptor in both sterile and nonsterile media. Ribulose diphosphate carboxylasewas stable throughout the exponential growth phase and declined slowly onlyafter cells reached the stationary phase. Reversible inactivation of the carbox-ylase occurred in extracts as a result of bicarbonate omission. The enzyme was

purified 32-fold with excellent recovery of a preparation which was 50 to 60%pure by the criterion of polyacrylamide gel electrophoresis. This purified prepa-

ration catalyzed the fixation of 1.25 umoles of CO2 per min per mg of proteinat pH 8.1 and 30 C, and the molecular weight of ribulose diphosphate carbox-ylase was approximately 350,000 daltons. A striking biphasic time course ofCO2 fixation that was independent of protein and ribulose diphosphate concen-

tration was observed. The optimal pH of the enzyme assay was fairly broad,ranging from 7 to 8.2. Kinetic dependence upon bicarbonate, ribulose diphos-phate, and Mg2+ was characterized and indicated that bicarbonate and Mg2+must combine with enzyme prior to addition of ribulose diphosphate. Anti-serum to ribulose diphosphate carboxylase from Hydrogenomonas eutropha was

only slightly inhibitory when added to the enzyme from T. denitrificans, andthe mixture did not precipitate. Cyanide (4 x 10-5 M) gave 61% inhibition ofthe enzyme from T. denitrificans. Ribulose diphosphate carboxylase in extractsof H. eutropha, H. facilis, Chromatium D, Rhodospirillum rubrum, and Chlo-rella pyrenoidosa were also inhibited to varying extents by cyanide and anti-serum to the H. eutropha enzyme.

Aside from the properties of pure ribulosediphosphate carboxylase (13, 14) from the hy-drogen bacteria, little is known about the en-zyme from other chemosynthetic bacteria. Fur-ther information about this enzyme, whichcatalyzes most autotrophic carbon dioxide fix-ation, may shed light on the evolution of auto-trophs. Knowledge of the enzyme from a che-mosynthetic organism which grows anaerobi-cally would be of interest because such an or-ganism might represent a primitive mode ofchemoautotrophism.We now describe the purification and prop-

erties of ribulose diphosphate carboxylase fromThiobacillus denitrificans and make a compar-ison of some of these properties with the en-zyme from other chemosynthetic and photo-synthetic microorganisms.

MATERIALS AND METHODSChemicals. The following special reagents were

'Present address: Department of Biochemistry, Univer-sity of California, Berkeley, Calif. 94720.

commercial preparations: dibarium and tetrasodiumribulose-1,5-diphosphate, deoxyribonuclease I, ribo-nuclease A, bovine serum albumin-fraction V, cata-lase, urease, and thyroglobulin from Sigma ChemicalCo.; Sephadex G-200' and dextran blue 2000 fromPharmacia Fine Chemicals; streptomycin sulfatefrom Mann Research Laboratories; Na214CO, (20Ci/mole) from Nuclear-Chicago Corp.; fumarasefrom Boehringer Mannheim Corp.; bovine serumalbumin dimer from Pentex Inc.; Folin phenol re-agent from Scientific Supplies Co.; Nobel agar, Bac-toagar, and purified agar from Difco Laboratories;and lonagar no. 2 from Colab Laboratories.

Other organic and inorganic chemicals were re-agent grade. Barium salts were converted to sodiumsalts prior to use by treatment with a slight excess ofsodium sulfate and removal of BaSO4 by centrifuga-tion. Purity of chemicals as indicated by the vendorswas taken into account in calculating concentra-tions. Unless otherwise indicated, buffers and chemi-cals were adjusted to the specified pH at 25 C withsulfuric acid, hydrochloric acid, or sodium hy-droxide. Tris(hydroxymethyl)aminomethane (Tris)buffers of specified pH contained any or all of thefollowing components: T, 0.01 M Tris-sulfate; E,0.003 M ethylenediaminetetraacetic acid (EDTA); M,

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634 McFADDEN

0.01 M MgCl2-H2O; and B, 0.05 M NaHCOs. Thus,TEMB buffer contained all these components at thespecified concentrations.

Cultures and growth conditions. T. denitrificanswas obtained from American Type Culture Collec-tion. The growth medium was prepared in deionizedwater according to Vishniac and Santer (23), and thepH was adjusted to 7.0 with sulfuric acid. The0.001% FeSO4 of that medium was routinely re-placed with the trace metal ion solution (1%, v/v) ofWolin et al. (26) prepared in deionized water. Forthe sterile media, the Na2S2O. and the K2HPO4 plusNaHCOs were autoclaved separately. After cooling,the latter solution was bubbled aseptically for 20min with CO2 before combining all ingredients.

For nonsterilized culture media, deionized waterwhich had been filtered through a Barnstead organicremoval filter to remove most organic material wasused to prepare fresh reagents. Unstirred liquid cul-ture in large carboys reached maximal growth in 2.5to 3 days at 25 to 28 C. Extent of heterotrophic con-tamination was periodically checked by streaking onagar containing 1% yeast extract. The purity of stockcultures of T. denitrificans was examined bystreaking upon the liquid culture medium solidifiedwith 1.7% purified agar. Growth occurred in approxi-mately 2 weeks at 25 C in desiccators evacuated andfilled to 50mm of CO2 pressure.

Cultures of Hydrogenomonas eutropha and H. fa-cilis were grown at 25 C essentially as described byRepaske (19). Cultures of Hydrogenomonas Hl andH16 obtained from Erwin Schuster were maintainedunder the same conditions.

Rhodospirillum rubrum standard, obtained fromJune Lascelles, was grown at 25 C in the medium ofGoodwin and Osman (10) in 1-liter standing cul-tures. Strictly anaerobic conditions were not neces-sary. Cultures were harvested in maximal yield in 2to 3 days. Stock cultures were stored in 1.7% agarstabs of the same medium. Rhodopseudomonas cap-sulatus from American Type Culture Collection wasmaintained in stab cultures on the medium ofGoodwin et al. (9) and grown in the same mediumfor 1.5 to 2 days in 1-liter standing cultures at 25 C.Cultures and extracts of Chromatium D as well as aculture of Chlorella pyrenoidosa were obtained fromRon Hurlbert. C. pyrenoidosa was grown at 25 C asdescribed by Krauss (15). All of the above photoauto-trophic cultures were grown under fluorescent lamps(15 w) at a distance of 6 to 15 inches. Immediatelyafter harvesting, cells were washed twice with TMBbuffer and used directly or frozen at -20 C for fu-ture use.

Cell-free preparations. Freshly harvested orfrozen cells of T. denitrificans or other organismswere suspended in TMB buffer, pH 7.9 (4 C), at aratio of 1 g of wet, packed cells per ml of buffer.Large volumes of the suspensions were ruptured at 2C at 15,000 to 25,000 psi with a French cell. Smallvolumes of suspensions of cells were ruptured at 2 Cwith a Biosonik probe-type oscillator at full power.Pressure-ruptured suspensions of T. denitrificanswere sometimes digested prior to centrifugation for30 min at 4 C with 1 mg of deoxyribonuclease, 1 mgof ribonuclease, or 1 mg of both per 50 g of original

AlND DENEND J. BACTERIOL.

wet, packed cells. Sonically treated or pressure-rup-tured suspensions of all organisms were centrifugedfor 1 hr at 105,000 x g, followed by dialysis at 4 Cfor 12 hr in TEMB buffer, pH 7.9. For T. denitrifi-cans, this dialyzed supernatant fraction derived fromuntreated extracts is denoted S105, whereas thesame fractions from ribonuclease- or deoxyribonu-clease-ribonuclease-treated extracts will be specifiedas R-S105, and DRS105, respectively.Enzyme assays. One unit of ribulose diphosphate

carboxylase catalyzed the fixation of 1 mmole ofCOJmin at 30 C under the conditions specified.Normally the reaction was initiated by the additionof 0.25 volume of ribulose diphosphate in water to1.0 volume of a solution that had been preincubated5 min at 30 C. The final mixture at pH 8.1 contained0.2 to 8 milliunits of ribulose diphosphate carboxyl-ase, 0.064 M Tris-sulfate, 0.02 M MgCl2, 0.02 MNal4CO, and 0.001 M ribulose diphosphate. Afterincubation for 5 min at 30 C with intermittent shak-ing, the reaction was terminated with 0.4 volume of60% trichloroacetic acid, and a sample was removedand placed in a scintillation vial for 2 hr to permitthe liberation of excess 14CO2. Radioactivity wasthen assayed (5) and corrected for slight fixationoccurring in the absence of ribulose diphosphate.Enzyme activity was estimated by consideration ofthe efficiency of counting "4C-benzoic acid stand-ards under identical conditions. Specific activity isexpressed as milliunits of enzyme per milligram ofprotein (16).

In some cases in which bicarbonate had been re-moved by dialysis from the buffer-containing en-zyme, bicarbonate was added to initiate the reaction.Ribulose diphosphate carboxylase during

growth of T. dentrificans. Six flasks, all containingidentical media and inocula of T. denitrificans, weregrown in 2-liter standing cultures at 25 C. At inter-vals, one flask was withdrawn, and a 25-ml sampleremoved. After the turbidity was measured, thesample was transferred to a tared membrane filter(0.45 gm pore size; Millipore Corp.), which was thendried and weighed. The filtrates were used to deter-mine pH. The remaining cell mass was centrifuged,washed, and frozen immediately. For an additionalset, the cultures were stirred very slowly but other-wise treated as described. The frozen cell masseswere thawed and broken, and the specific activitieswere then determined.

Immunological experiments. The immunodiffu-sion technique was employed to detect precipitinreactions between rabbit antiserum against ribulosediphosphate carboxylase from H. eutropha and theenzyme in crude extracts of other autotrophs. Diffu-sion was carried out on microscope slides overlayedwith agar as described by Campbell et al. (6), exceptthat the agar solution contained 0.2 ml of 0.2%trypan blue-0.2% Merthiolate per 100 ml.A rabbit was immunized by subcutaneous injec-

tions at 1-week intervals with 200 to 400 Ag of pureribulose diphosphate carboxylase from fructose-grown H. eutropha (13) in the presence of methyl-ated bovine serum albumin and Freunds adjuvant(Difco). After seven injections, the rabbit was bled;10 ml of the serum was fractionated with diethyl-

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RIBULOSE DIPHOSPHATE CARBOXYLASE

aminoethyl cellulose, and gamma globulins were pre-cipitated by addition of (NH4)2SO4 to 60% saturationat 2 C (6). After centrifugation, the pellet was dis-solved in 5 ml of borate-saline buffer (6) and dialyzedagainst the same buffer and finally against 0.05 MTris, pH 7.5 (25 C), containing 0.015 M NaCl. Thegamma globulin fraction was then stored at -20 Cuntil used.The extent of cross-reactivity between antibodies

and the various ribulose diphosphate carboxylaseswas also examined in tests of inactivation by thegamma globulin fraction of the antiserum. The car-boxylase in extracts from H. eutropha, strain Hi orH16, was incubated with increasing amounts of thegamma-globulin fraction for 30 min at 30 C beforeassay by the normal procedure. A single concentra-tion of that fraction was tested against extracts fromT. denitrificans, Chromatium D, R. rubrum, H. fa-cilis, and C. pyrenoidosa.Cyanide inhibition. The possibility of inhibition

of ribulose diphosphate carboxylase by cyanide wastested for the enzymes from T. denitrificans, H. eu-tropha, H. facilis, Chromatium D, R. rubrum, and C.pyrenoidosa. Ordinarily, cyanide was preincubatedwith the enzyme plus 0.05 M Na14CO3 and 0.003 MMg2+ for 3 min before being mixed with other com-ponents, and the reaction was initiated with ribulosediphosphate in accord with the normal assay proce-dure.

Fractionation of the enzyme from T. denitrifi-cans. The dialyzed supernatant fraction (RS105,or DRS105) of T. denitrificans was adjusted to 10mg of protein/ml with TMB (pH 7.9, 4 C). In somecases a 10% solution of streptomycin sulfate, pH 7.0,was added dropwise to R S105 with constant stir-ring to a final concentration of 0.75 mg of strepto-mycin sulfate/mg of protein. After 40 min, the com-plexed deoxyribonucleic acid (DNA) was sedimentedat 29,000 x g for 15 min. To the supernatant fraction,SM10, or to DR-.S105, solid ammonium sulfate wasadded slowly at 2 C in increments to 48% saturation(45% at 25 C) followed by pelleting of the solid ma-terial at 29,000 x g for 15 min. Subsequently, thesupematant fraction was adjusted to 59% saturation(55% at 25 C), and the pellet was collected and dis-solved completely in 250 ml of TMB, pH 7.9, at 4 C(SAS-1).

Fraction SAS-1 was then applied to a SephadexG-200 column bed (2.5 by 40 cm), equilibrated at 4 Cwith TMB, pH 7.9 (4 C), and eluted at a flow rate of5 ml/hr and a pressure head of 6 cm. The effluentfrom the column was analyzed for ratios of absorb-ancy at 280 and 260 nm, specific activities, and thepooled, specific-activity peaks (SG-200) were reas-sayed. This product was again subjected to fraction-ation with solid ammonium sulfate as described ear-lier (SAS-2).Enzyme purity. Ribulose diphosphate carboxylase

preparations were examined for purity by polyacryl-amide disc gel electrophoresis (7). Running gels atpH 9.5 were used, omitting stacking and spacer gels.Approximately 50 ug of protein in 20 uliters of 30%sucrose-buffer solutions was applied to the top of thegels. A current of 2 ma/gel was applied through thecolumns at room temperature until the tracking dye

(bromothymol blue) approached the end of the gels(1.5-2.5 hr). The gels were extruded from the tubes,stained for 5 min in aniline black (0.5% in 7% aceticacid), and destained in successive washes of 1.5%acetic acid. The gels were then scanned at 550 nmwith a Gilford 2400 spectrophotometer with a 2410linear transport operating at 1 inch/0.5 min.pH dependence. To determine the effect of pH on

the activity of ribulose diphosphate carboxylase fromT. denitrificans, assays were performed by thenormal procedure, except that incubation mixturescontained instead the following buffering species at0.06 M and the pH values indicated: citrate, 4.7, 5.4,5.7; histidine, 6.0, 6.4, 8.9, 9.3; imidazole, 6.8, 7.0,7.4; and Tris, 7.6, 7.8, 8.0, 8.2, 8.4, 8.65. All adjust-ments in pH were made with HCl.Molecular weight. To measure the molecular

weight of ribulose diphosphate carboxylase in theSAS-2 fraction from T. denitrificans, the method ofAndrews (3) was used. The enzyme and standardswere chromatographed and eluted from a SephadexG-200 column at 4 C with 0.02 M imidazole con-taining 0.1 M KCl, 1 mM ,-mercaptoethanol, and 1mM EDTA (pH 7.3). The column (0.5 by 50.8 cm)had been equilibrated with the same buffer.

RESULTSGrowth and ribulose diphosphate content

of T. denitrificans. Growth was consistentlyobtained at 25 to 30 C in standing mass cul-tures of up to 50 liters with thiosulfate as elec-tron donor and nitrate as electron acceptor.Stock cultures were routinely stored aftergrowth in 30- to 50-ml glass-stoppered orscrew-capped vials filled with Baalsrud's me-dium (23). Growth response on plates of thesame medium solidified with agar was ex-tremely slow, perhaps in part due to tendencyof crystals to form in the medium. No growthwas observed on agar stabs nor did aerobicgrowth of T. denitrificans occur in the absenceof nitrate. Pure cultures of T. denitrificanstransferred to nonsterilized medium developedless than 1% heterotrophic contaminationthroughout exponential and early stationaryphase.The activity of ribulose diphosphate carbox-

ylase in T. denitrificans was stable throughoutthe exponential growth phase (Fig. 1). A 50%loss in specific activity was observed well intostationary phase after growth for about 150 hr.Even after this loss in activity, the enzymewas stable to subsequent manipulation. Theage of the inoculum had no effect on the spe-cific activity, although it did have an effect onthe length of the initial lag phase. The de-crease in pH from 7.0 to pH 5.5 to 6 duringnormal growth did not affect the resultantspecific activity, nor did the ambient tempera-ture range (25-38 C) at which the organism was

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80

II 60

y

> 40

2.

~R 20

40 60Hours

n

sr

UL)

0L

FIG. 1. Correlation of growth of T. denitrificansand specific activity of ribulose diphosphate carbox-ylase. A red filter was used for turbidity measure-ments with a Klett colorimeter.

grown affect the cell yields or specific activity.For large cultures, slow stirring of the growthmedium kept the cells from settling and hadlittle or no effect upon the specific activity ofthe enzyme.Sequence of substrate addition. The order

of addition of substrates to the enzyme pro-

foundly influenced the reaction rate. As illus-trated in Fig. 2, preincubation of crude or par-

tially purified enzyme with ribulose diphos-phate (+Mg2+) resulted in lower activity thanpreincubation of the enzyme with bicarbonate(+Mg2+). Furthermore, increased preincuba-tion with ribulose diphosphate had an inhibi-tory effect upon the crude enzyme. When dia-lyzed or diluted in the absence of bicarbonate,e.g., in TM (pH 7.9, 4 C), the ribulose diphos-phate carboxylase was partially inactivated.However, this effect was reversed in 5 to 10min at 30 C by readdition of bicarbonate atnormal concentrations.Cyanide inhibition of ribulose diphos-

phate carboxylase from various autotrophs.Reversible inhibition of the spinach leaf en-

zyme by low concentrations of cyanide hasbeen observed by Lane and co-workers (24),who also implicated a bound cupric ion as thesite of inhibition (25). Similar low concentra-tions of cyanide were found to effectively in-hibit ribulose diphosphate carboxylases frombacteria and algae (Table 1). Except for 54%inhibition observed with the enzyme from R.rubrum, very slight inhibition occurred at 4 x10-6 M cyanide (not shown).Further experiments were done in which the

cyanide was preincubated with the enzyme,with each substrate, or with both enzyme and

one substrate before initiation of the reactionwith the second substrate. The greatest inhibi-tion was apparent for those cases in which thecyanide was preincubated with the enzymeand ribulose diphosphate together. This is con-

sistent with the results of Wishnick and Lane(24) who suggested that the cyanide reactswith the enzyme only in the presence of ribu-lose diphosphate.Immunological studies. Using antiserum to

0

x_4Purified RDPC

X I

0 3 6 9Preincubotion Time (min)

FIG. 2. Effect of preincubation at 30 C of crudeenzyme (DR - S105) with HCOS- (-) and ribulosediphosphate (0) and 50%o pure enzyme (SAS-2; see

Table 3) with HCO.- (-) and ribulose diphosphate(0); 0.76 milliunits of crude and 3 milliunits of 50%pure enzyme were used per assay.

TABLE 1. Inhibition of ribulose diphosphatecarboxylases by cyanide

Per cent inhibitiona

Organism 4 x 10- I'M 4 x 10-4 MCN- CN -

Thiobacillus dentrificans 61.4 91.7Hydrogenomonas eutropha 28.2 73.8H. facilis ................. 14.0 72.0Chromatium D ............ 10.1 73.9Rhodospirillum rubrum .... 64.6 91.8Chlorella pyrenoidosa ...... 6.6 57.6

a Under normal assay conditions, cyanide (CN-)was preincubated with the enzyme Mg2+-HCO3 mix-ture for 3 min prior to initiation of the reaction withribulose diphosphate. At 4 x 10-' M and 4 x 10-4 M,CN concentrations were, respectively, 6.7 and 67% ofthe ribulose diphosphate concentrations in the assay.Enzyme specific activities were 39, 10, 16, 46, 53,and 34 from the sources shown from top to bottom.

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pure ribulose diphosphate carboxylase from H.eutropha, no cross-reactions by the Ouchter-lony double-diffusion technique were observedwith the enzyme from sources other than Hy-drogenomonas. Included in this series were

Hydrogenomonas strains Hi and H16, T. de-nitrificans, Chromatium D, and autotrophicallyand fructose-grown H. eutropha and H. facilis.Identity reactions were observed for the en-

zyme from autotrophically grown H. eutrophaand strain Hi with that from fructose-grownH. eutropha (Fig. 3). A very faint precipitinband was apparent for the enzyme in extractsof strain H16. Inactivation by antibodies was

then characterized for the enzyme from H.eutropha and strains Hi and H16 (Fig. 4). Asevident, the titration curve for Hydrogeno-monas Hi followed more closely that of H.eutropha, although initially it did not drop as

sharply. The titration curve for strain H16 dif-fered significantly, and its trend towards aplateau at 2 to 3 ,uliters of the gamma globulinfraction was reproducible. We have no expla-nation for this. Inhibition of 50% of the en-

zymic activity from H. eutropha and strain Hiwas obtained with 2 to 3 gliters of the anti-body fraction, as opposed to the strain H16preparation which required about 6 gliters.Serum from an unimmunized rabbit had noeffect upon the enzymic activities.

Single concentrations of the antibody frac-tion were tested against ribulose diphosphatecarboxylase from other sources as indicated inTable 2. With the exception of the enzymefrom strain Hi and H. eutropha, loss of ac-

FIG. 3. Double-diffusion precipitin reactions ofthe gamma-globulin fraction of antiserum to theHydrogenomonas eutropha enzyme with crude ex-

tracts of H. eutropha and Hydrogenomonas Hl.Center wells contained antibodies to pure ribulosediphosphate carboxylase from fructose-grown H. eu-

tropha. The lower case letter is flanked by the speci-fied extract. For display A these are: (a) fructose-grown H. eutropha and (b) autotrophically grown H.eutropha; and for display B: (a) autotrophicallygrown Hydrogenomonas Hl and (b) fructose-grownH. eutropha.

100

>Z 80

C 60._

E

M 40

0

%-- 20

0 L_0 2 4 6 8

y-Globulin Soin. (FIL)FIG. 4. Inactivation of ribulose diphosphate car-

boxylase by the gamma globulin fraction of anti-serum to the enzyme from Hydrogenomonas eu-tropha at 30 C. Assay mixtures initially contained0.65, 0.98, and 0.95 milliunits of ribulose diphos-phate carboxylase from H. eutropha (A), Hydrogeno-monas Hl (0), and H16 (U), respectively.

TABLE 2. Antibody inactivation of ribulosediphosphate carboxylases

Source of enzymea Remainingactivity()

Hydrogenomonas eutropha ...... .... 25.8Hydrogenomonas Hi ........ ....... 27.2Rhodospirillum rubrum ............. 47.2Hydrogenomonas H16 ....... ....... 60.4Chlorella pyrenoidosa ....... ....... 69.9H. facilis .......................... 70.7Chromatium D ..................... 78.7Thiobacillus denitrificans ...... ..... 93.3

a Under normal assay conditions, the enzyme waspreincubated with 5 gliters of antibody before initi-ating assay. Milliunits per assay ranged from 0.64 to1.0 for Hydrogenomonas Hi and H16 and H. eu-tropha and 0.78 to 2.76 for the enzyme from re-maining sources.

tivity ranged from 7 to 53%. The enzyme fromR. rubrum, the closest to strain Hi and H. eu-tropha with respect to lost activity, still hadnearly twice the activity of the latter. Theenzyme from strain H16 again appeared to bedistinctly different from the enzyme from ei-ther strain Hi or H. eutropha.

Purification and stability of ribulose di-phosphate carboxylase from T. denitrifi-cans. Table 3 summarizes the best purifica-tion procedure for the enzyme from T. denitri-

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McFADDEN AND DENEND

ficans. Starting material was 200 g of wet,packed cells. This procedure resulted in a sev-enfold increase in specific activity and a 223%recovery of total enzyme units upon ammo-nium sulfate fractionation. Previous trials hadalso yielded increases in total units with strep-tomycin sulfate treatment. Striking increasesin total enzyme activity have also been ob-served during fractionation of ribulose diphos-phate carboxylase from H. facilis and H. eu-tropha (13) and possibly indicate the removalof a natural nondialyzable macromolecularinhibitor.The fractionation procedure described in

Table 3 included only a small portion of theSAS-1 fraction (280 mg of the 2,286 mg of pro-tein in SAS-1) which was applied to the Seph-adex G-200 column. When treatment ofR-S105 with streptomycin to yield SM10 wassubstituted for the first step in Table 3, twopeaks of enzyme activity were obtained uponchromatography on Sephadex G-200. The first,which eluted directly after the void volume,contained 22% of the activity and had a ratioof absorbancy at 280 and 260 nm of 0.75. The

TABLE 3. Purification of ribulose diphosphatecarboxylase from T. denitrificans

Total Enzyme Total Per PurlfrFraction protein specific activity cent cation

(mg) activity (units) yield

DR, S105 7,080 11 77,880 (100) (1)SAS-1 2,286 76 173,700 223 7SG-200 988 156 154,600 199 14SAS-2 310 344 106,700 137 32

second contained the rest of the enzyme andhad an absorbancy ratio in excess of 1.1. Incontrast, fractionation as described in Table 3resulted in a single activity peak with a smallshoulder on each side (Fig. 5). Only those frac-tions with specific activities greater than 150were pooled for further manipulation.The final ammonium sulfate precipitate

(SAS-2) was 50 to 60% pure (Fig. 6). This prep-aration represents a 32-fold purification andhad a final specific activity of 344. As will be-come evident, this specific activity is actuallyabout 1,250 when a revised assay procedure isused.The results for the isolation protocols de-

scribed were obtained under conditions inwhich the ribulose diphosphate carboxylasewas always maintained in the presence of 50mM HCO3- and 10 mM Mg2+ for protectivepurposes. The enzyme, however, was found tobe quite stable to freezing at -20 C in all frac-tions even when bicarbonate and Mg2+ hadbeen omitted from the 0.01 M Tris buffer, pH7.9.

In tests of isoelectric precipitation, morethan 80% of the enzyme activity precipitatedbetween pH 5.0 and 5.5. The recovered en-zyme had a twofold higher specific activity,but because the overall recovery was poor(39%), isoelectric precipitation was abandonedas a purification step.Optimal pH for the enzyme from T. deni-

trificans. The pH optimum was broad andbetween the limits of 7 and 8.2. Correctionswere made for the decreasing concentration ofH14CO03- at lower pH values.Enzyme assay. The rate of fixation cata-

0

C0co

8

Ct

0

Lj

.E2

8

0)

E.E4

8

.0v0

- -

20 40 60 80 100Fraction Number

FIG. 5. Elution profile for chromatography upon Sephadex G-200 of fraction SAS-1 from Thiobacillus de-nitrificans.

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VOL. 110, 1972 RIBULOSE DIPHOSPHATE CARBOXYLASE 639

0.32 to 0.60 mM were treated in the Hill equa-12 tion as modified by Atkinson (4), the interac-

tion coefficient, n, was 2.0. The substrate con-centration yielding 0.5 of the maximal veloc-

I.0O ity, (RDP) 0.5 (4), was 0.12 mM.Saturation of the enzyme by bicarbonate

E (Fig. 9) was analogously abnormal. For data in0 0.8 _ the range of 4 to 28 mM, n was 1.6. The value0 of (HCO3-)o.5 was 14 mM.

06-

c] ,*~~~~~~~~~~~~~~~~~~~~~~a

oL04- o0

LL 40

0 2 3 4 5 6 0Distonce Along Gel (cm.)

a,

FIG. 6. Scan at 550 nm of polyacrylamide gel Eelectrophoregram of ribulose diphosphate carbox- o 20ylase (fraction SAS-2) from Thiobacillus denitrifi- Jcans.

lyzed by ribulose diphosphate carboxylase 01from T. denitrificans demonstrated a linear 0 2 4 6dependence on enzyme concentrations up to Time (Mi10 milliunits for a 5-min assay. The time

F T e (CO,

)course of fixation is shown in Fig. 7. As evi- FIG. 7. Time course of CO2 fixation catalyzed bydent, biphasic time courses of fixation were ribulose diphosphate carboxylase (fraction SAS-2)daent,bifor assay mixtures containing 15, 3 from Thiobacillus denitrificans at pH 8.1, 30 C. Forapparent for ass of ues Thetining up- the assays, 15 milliunits (A), 3 milliunits (U) and 1.5and 1.5 milliunits of enzyme. The initial up- milliunits (0) were used.take of HCO3- remained proportional to thetime for less than 1 min. The second phaseappears to be linear between 2 to 6 min ofassay time. The specific activity calculatedfrom the lower portion of the curve is 1,250milliunits/mg of protein, which is two- to 66-threefold higher than that estimated at thenormal assay time of 5 min. The biphasic na-ture was unaffected by substitution of histi- 'CU_dine for Tris buffer or by preincubation of ri- N 4bulose diphosphate in Tris buffer.Kinetic studies with the enzyme from T.

denitrificans. Normal substrate dependence IV

was exhibited at higher protein concentrations g 2(17 milliunits per assay). However at that en-zyme concentration, depletion of ribulose di- Zphosphate ranged from 60 to 43% for 1.2 x 10-i 00and 1.2 x 10- 3 M, respectively. Substrate 0 2 4 6 8 10dependence at a lower enzyme concentration (RDP) x 104(2 milliunits per assay) is shown in Fig. 8. FIG. 8. Dependence of rate upon ribulose diphos-Double reciprocal plots showed two linear phate (RPD) concentration for the enzyme (fractionphases of different slope. When data for ribu- SAS-2) from T. denitrificans. Assay mixtures at pHlose diphosphate concentrations in the range of 8.1, 30 C, contained 6 milliunits.

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2

c-,0,cn

0E0c-z

0 20 40

mM of HCO3FIG. 9. Saturation of ribulose diphosphate carbox-

ylase in fraction SAS-2 by bicarbonate at pH 8.1, 30C.

The enzyme in fraction SAS-2 from T. deni-trificans showed an absolute dependence uponMg2+ but at 30 C, pH 8.1, was saturated at 0.1mM, a concentration well below the Km forribulose diphosphate carboxylase from othersources (11).Molecular weight. The apparent molecular

weight of the enzyme in fraction SAS-2 fromT. denitrificans was 350,000 (Fig. 10) as elutedfrom Sephadex G-200 at a protein concentra-tion of 2.5 mg/ml.

DISCUSSIONLevels of ribulose diphosphate carboxylase

are invarient from early logarithmic growththrough early stationary phase during growthof T. denitrificans, and the enzyme is stable inextracts in the absence of bicarbonate. In con-

trast, the enzyme is unstable in extracts fromfructose-grown H. eutropha in the absence ofbicarbonate (12). Moreover, in Hydrogeno-monas the enzyme is degraded or inactivatedupon depletion of fructose, although the en-

zyme is stable in starved cells of autotrophicorigin (12). Of interest in this connection are

the present studies which demonstrate immu-nological identity of the enzyme from fructose-grown and autotrophically grown H. eutropha.Enzyme of 50 to 60% purity has been ob-

tained in four steps from T. denitrificans inexcellent yield. Of added interest is the factthat this organism can be grown in mass cul-ture almost free from heterotrophic contami-nation without sterilization of the medium.

Thus both the ease of culture and enzyme pu-rification suggest that T. denitrificans will bean excellent future source of ribulose diphos-phate carboxylase. The enzyme from T. deni-trificans has a pH optimum that extendssomewhat further towards neutrality than istypical (11). The data support an ordered reac-tion in which ribulose diphosphate reacts witha complex containing CO2 and Mg2+ as withother ribulose diphosphate carboxylases (13,17, 18). Although the enzyme is saturated atunusually low concentrations of Mg2+, ap-parent Km values for bicarbonate and ribulosediphosphate are normal (11). Of interest arethe interaction coefficients greater than oneobserved in certain concentration ranges ofboth substrates at saturating Mg2+. Thusbinding or turnover of both ribulose diphos-phate and bicarbonate is cooperative undercertain conditions. The effect of pH and Mg2+upon this cooperativity was not examined inthe present work. Homotropic interactions forbicarbonate with the spinach enzyme (22) andfor ribulose diphosphate with the rhodopseudo-monad enzyme (20) are apparent at certainMg2+ concentrations and pH values.

In the present studies of ribulose diphos-phate carboxylase from T. denitrificans, aburst of CO2 fixation has been discovered intimes of a minute or less. After this burst, therate falls to less than half of the original ve-locity. The burst could be masked in studies ofincorporation at later intervals and may have

' >r

4x104 10- 5X10< 10

Molecular Weight

FIG. 10. Plot of (Ve - V0)/V. versus molecularweight (logarithmic scale) for proteins chromato-graphed on Sephadex G-200. V, VO, and V. are elu-tion, void, and total minus void volumes, respec-tively. BSA and RDPC are bovine serum albuminand ribulose diphosphate carboxylase, respectively.

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RIBULOSE DIPHOSPHATE CARBOXYLASE

been overlooked in other laboratories. In thisconnection, Rabin and Trown (18) proposed anordered release of the two phosphoglyceratemolecules from enzyme, one being very fast,and the second very slow and rate limiting,possibly as a consequence of covalent bindingat a second site. Perhaps approach to satura-tion at the second site retards the fast rate.Whatever the explanation, our results raise thepossibility that in vivo rates of CO2 fixationhave been underestimated if ribulose diphos-phate carboxylase is less than saturated insitu.

In the present studies, the molecular weightof the enzyme from T. denitrificans has beenestimated by gel filtration with the usual as-sumption that the standards and enzyme havesimilar densities and shapes (3). The value of350,000 is much higher than that for the en-zyme from R. rubrum (1, 2), about equal tothat for Rhodopseudomonas (1, 2), and some-what more than half that for Hydrogenomonas(14), Chromatium (2), blue-green (2) and greenalgae (1, 2, 21), and higher plants (11). Al-though these molecular-weight trends are notsystematically reflected in our studies of im-munological relationships or cyanide inhibi-tion, more research is clearly needed in theseareas.

Davis et al. (8) have recently proposed a re-classification of H. eutropha into the genusAlcaligenes largely because it is peritrichouslyflagellated in contrast to other species of Hy-drogenomonas, which they proposed to reclas-sify into the genus Pseudomonas. Hydrogeno-monas Hi and H16 were designated as repre-sentative strains of H. eutropha. On the basisof the present limited immunological data forribulose diphosphate carboxylase, the enzymefrom strain H16 is quite different from those ofstrain Hi and H. eutropha. In fact it is moresimilar to that from H. facilis. It seems prema-ture to reclassify organisms which share sev-eral important and distinctive biochemicalproperties into different genera until more in-formation is available and a more quantitativebasis for comparison is established. In the finalanalysis this will depend upon the degree ofhomology between genomes.

ACKNOWLEDGMENTSThis research was supported by Public Health Service

grant AM-14400 from the National Institute of Arthritis andMetabolic Diseases and by predoctoral fellowship GM-39114 from the National Institute of General Medical Sci-ences.We thank G. D. Kuehn for preparation of the antiserum

to ribulose diphosphate carboxylase and the derived gammaglobulin fraction.

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12. Kuehn, G. D., and B. A. McFadden. 1968. Factors af-fecting the synthesis and degradation of ribulose-1,5-diphosphate carboxylase in Hydrogenomonas facilisand Hydrogenomonas eutropha. J. Bacteriol. 95:937-946.

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17. Pon, N. G., B. R. Rabin, and M. Calvin. 1963. Mecha-nism of the carboxydismutase reaction.I. The effect ofpreliminary incubation of substrates, metal ion andenzyme on activity. Biochem. Z. 338:7-19.

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