Pyruvate Is a By-product of Catalysis by Ribulosebisphosphate

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					THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 15, Issue of May 25, pp. 9447-9452, 1991 Printed in U.S.A .

Pyruvate Is a By-product of Catalysis by Ribulosebisphosphate Carboxylase/Oxygenase*
(Received for publication, November 29, 1990)

T. John Andrew& and Heather Kane J.
From the Plant Environmental Biology Group, Research School Biological Sciences, Australian National University, of . ” Canberra ACT 2601, Australia

Pyruvate is a minor productof the reaction catalyzed ase (EC4.1.1.39) is fundamental to almost all forms of photoby ribulosebisphosphate carboxylase/oxygenase from or chemolithotrophic life. However, despite presumably inspinach leaves. Labeled pyruvate was detected, in ad- tense selective pressure to maximize its catalytic efficiency, dition to the major labeled product, 3-phosphoglycer- this enzyme seems poorly adapted to its task. Its feeble V,,,/ ate, when I4CO2was the substrate. Pyruvate produc- K,,, ratio necessitates that plants allocate as much as half of tion was also measured spectrophotometrically in the their soluble leaf protein to this one enzyme. Furthermore, its presence of lactate dehydrogenase and NADH. The difficulty in distinguishing between C02 andO2 as substrate K , for COzof the pyruvate-producing activity was encumbers photosynthetic organisms with the wasteful proc12.5 MM, similar to theCOz affinity of the 3-phospho- ess of photorespiration (reviewed by Andrews and Lorimer, glycerate-producingactivity. No pyruvatewas de- 1987). tected by the coupled assay when ribulose 1,5-bisphosThe catalytic sequence of ribulose-Pn carboxylase involves phate was replaced 3-phosphoglycerateor when the by 1). carboxylase was inhibited by the reaction-intermedi- several enzyme-bound intermediates (Scheme Calvin’s sixcarbon, carboxylated intermediate (Calvin, 1956) (shown in ate analog, 2‘-carboxyarabinitol 1,5-bisphosphate. free gemdiol forms Therefore, pyruvate was not being produced from 3- Scheme 1in both the ketone and hydrated, as I1 and 111) hasprovenstable enough to be isolatedin phosphoglycerate by contaminant enzymes. The ratio quantity, and its reactions in solution and on the active site of pyruvate produced to ribulose bisphosphate conhave “C sumed a t 2 5 was 0.7%,and this ratio was not altered been characterized (Schloss and Lorimer, 1982; Pierce by varying pH or COz concentration or substituting et al., 1986; Lorimer et al., 1986; Andrews and Lorimer,1987). by Mn”+for Mg2+as the catalytically essential metal. The The five-carbon enediol(ate) (I), the species to which the ratio increased with increasing temperature. Ribulose- gaseous substrates add, has been revealed as a very unstable bisphosphate carboxylases from the cyanobacterium species which eliminates the C-1 phosphate moiety very rapSynechococcus PCC 6301 and the bacterium Rhodo- idly when released from the active site (Jaworowski et al., spirillum rubrum also catalyzed pyruvate formation 1984). The intermediacyof this species was further supported and to the same extent as the spinach enzyme. When recently by observations thatisolated ribulose-Pncarboxylase the reaction was carried out in ‘HzO, the spinach car- slowly epimerizes ribulose-P2 to produce the potent inhibitor, boxylase increased the proportion of its product par- D-xyhlOSe 1,5-bisphosphate, leading to progressive inhibition titioned to pyruvate to 2.2%. These observations pro- during catalysis. This epimerization results from stereochemvide evidence that the C-2 carbanion form of 3-phos- ically incorrect reprotonation of the enediol (Edmondson et phoglycerate is an intermediatethe in catalytic sequence of ribulose-bisphosphate carboxylase. Pyru- al., 1990a-d). The final intermediate shown in Scheme 1 (IV) @ vate is formed by elimination of a phosphate ion from is the C-2 carbanion (or aci-acid) of P-glycerate. Following Jaworowski et al. (1984), we use the term “aci-carbanion” to a small portionof this intermediate. describe this resonant species. Evidence for the involvement of this species is less direct than for the earlier two intermediates. The strong isotope effect associated with the attachThe reactioncatalyzed by ribulose-Pn’carboxylase-oxygen- ment of solvent tritium to the C-2 P-glycerate (Hurwitzet of al., 1956; Simon et al., 1964; Fiedler et al., 1967; Saver and * The costs of publication of this article were defrayed in part by Knowles, 1982) suggests the involvement of an intermediate the payment of page charges. This article must therefore be hereby whose protonation allows competition between ‘H and ‘H. marked “advertisement” in accordance with 18 U.S.C. Section 1734 Jaworowski et al. (1984) were unable to detect the expected solely to indicate this fact. 2 To whom allcorrespondenceandreprintrequestsshould be breakdown product of such an aci-carbanion (pyruvate) after addressed: Plant Environmental Biology Group, Research School of acid quenching during turnover. However, their method did Biological Sciences, Australian National University, P. 0. Box 475, not have the sensitivity necessary to detect the small traces Canberra ACT 2601, Australia. Tel.: 61-6-249-5072; Fax: 61-6-248of such a breakdown product which might be expected if the 9995. aci-carbanion did not accumulate high levels on theenzyme to I The abbreviations used are: ribulose-Pp, D-ribulose 1,5-bisphos(see “Discussion”). phate;ribulose-P2 carboxylase, ribulose-bisphosphate carboxylase/ The epimerization of ribulose-Pn that ribulose-P2 carboxoxygenase; carboxypentitol-P2, unresolvedisomeric mixture of 2’carboxy-a-arabinitol 1,5-bisphosphate and 2’-carboxy-~-ribitol 1,5- ylasecatalyzes (Edmondson et al., 1990d)shows that the bisphosphate; 3-keto-carb~xyarabinitol-P~, 2’-carboxy-3-keto-~-ara-active site is unable to totally prevent unwanted side reactions binitol 1,5-bisphosphate; P-glycerate, 3-phospho D-glycerate; Hepps, associated with the highly reactive enediol intermediate (I). N-2-hydroxyethylpiperazine-N’-3-propanesulfonic Hepes, N-2acid; hydroxyethylpiperazine-N’-2-ethanesulfonic acid Bicine, N,N-bis(2- We reasoned that, if the aci-carbanion (IV)was a significant hydroxyethy1)glycine; Pipes, piperazine-N,N’-bis(2-ethanesulfonic intermediate, it might also be subject to abortive side reactions. This intermediatewould be expected to be subject to p acid); Ches, 2-(cyclohexylamino)ethanesulfonicacid.

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9447

9448

Pyruvate Production by Ribulose-P2 Carboxylaseloxygenase

of I4CO2were chromatographed on a Bio-Rad Aminex HPX87H column (Fig. 1).In addition to the expected major radioactive peak of phosphoglyceric acid, a small peak of radioactivity was observed which coeluted with the UV peak due to the pyruvic acid internal standard. The identity of this peak as pyruvicacidwasconfirmed by the observation that it disappeared, and the radioactivity moved to coelute with lactic acid, when lactate dehydrogenase and NADH were present during the carboxylation reaction.very small peakof radioA activity coeluted with glyceric acid under both conditions. Presumably itwas the resultof a trace of phosphatase present in the ribulose-P2 carboxylase preparation. Pyruvate Production Is an Intrinsic Property of the Ribulose-P2 Carboxylase Reaction-Pyruvate production was observed spectrophotometrically in a continuous assay by coupling to the lactatedehydrogenase reaction (Fig. 2). Activity proceeded in an approximately linear fashion untilribuloseP, became limiting, and it eventually ceased when ribuloseEXPERIMENTAL PROCEDURES P, was exhausted. The extentof the reaction corresponded to Materials-Ribulose-P, carboxylase was purified from spinach less than 1%of the ribulose-P, initially present (Table I) leaves and ribulose-P? was synthesized as previously described (Ed- because most of the ribulose-P2 was being converted t o P mondson et al., 1990a). Synechococcus PCC 6301 ribulose-P2 carbox- glycerate,which issilentinthis assay. No pyruvate was ylase was purified from extracts of Escherichia coli coexpressing the rbcL and rbcS genes onplasmid p S H l (Andrews, 1988) by the produced when P-glycerate was substituted for ribulose-P,, procedure of Andrews and Ballment (1983). Rhodospirillurn rubrum showing that the pyruvate was not being produced from P ribulose-P, carboxylase was purified from extracts of E. coli express- glycerate by contaminating enzymes in the ribulose-P2 caringthe rbcL gene onplasmid pRRl (Morel1 et al., 1990) a by boxylase or lactate dehydrogenase preparations. Pretreatment combination of (NH,),SO, fractionation (30-60% saturated fraction of the ribulose-P2 carboxylase with carboxypentitol-Ps, which taken),anion-exchangechromatographyon a Mono Q HR 10/10 containsthestronglyinhibitoryanalog of thesix-carbon column(PharmaciaLKB Biotechnology Inc.)with KC1 gradient 2'-carboxyarabinitol-P2,completely inelution, and gel filtration on a Pharmacia Superose12 H R 10/30 reaction intermediate, even column. Carboxypentitol-P, was synthesized according to Collatz et activated the pyruvate-producing reaction and, whenthe al. (1979) and stored in 50 mM Bicine-NaOH buffer, pH 9.0. Other inhibitor was added after ribulose-P,, pyruvate production enzymes were obtained from Sigma or Boehringer Mannheim. stopped promptly well before the ribulose-P, was exhausted. K , (COJ Measurement-The rate of pyruvateproduction was Whenpyruvateproductionandtotal14C0,fixation were measured at 25 "Cspectrophotometrically a t 340 nm. A solution measured in parallel identical reaction mixtures (Fig. 3), the containing 130 mM Hepps-NaOH, pH 8.3, 18 mM MgCL, 50 pM NADH, and 1 mM ribulose-P2 was madeC0,- and Oa-free byexhaus- time-courses for the two reactions were identical exceptfor a tive sparging with N2. After transferto a stopperedcuvetteand brief lag in the oxidation of NADH caused by the coupling further sparging, sparging wasdiscontinued andHCO,, bovine eryth- system used in the pyruvate assay. All of these data confirm rocyte carbonic anhydrase, and rabbit muscle lactate dehydrogenase that pyruvate must be produced as an integral part of the addedto 0.05-20 mM, 100 pg/ml, andapproximately 3 units/ml, respectively. Catalysis was then initiated by adding spinach ribulose- reaction mechanism of ribulose-P, carboxylase. Ratio of Pyruvate Production to Total Carboxylation-This P, carboxylase (final concentration of catalytic sites, 2.1 p ~ which ) had been preactivated in the presence of 19 mM MgCI, and 10 mM ratio may be calculated either from the extents of the two NaHCO,,. or reactions (Table I) from their initial rates (data not shown). Other Methods-Concentrations of spinach ribulose-P, carboxyl- Similar ratios were obtained using both methods. The ratio ase were estimated spectrophotometrically at 280 nm using the published absorption coefficient (Paulsen and Lane, 1966). Ribulose-P2 was approximately 0.7% and was remarkably unaffected by For spinach ribulose-P2 carboxconcentrations were measured spectrophotometrically usingan assay varying conditions (Table I). similar to that described for P-glycerate in the legend of Table I, ylase, varying pH between 6.4 and 9.1 was without effect, as except that 10 mMHCO: and 20 pg/ml spinach ribulose-P2 carbox- was varying theHCO, concentration between 0.5 and 10 mM ylase were also present. at pH 8.3. Substituting Mn2+ for Mg2+ as the catalytically essential metal also did not alter the ratio. The enzymes from RESULTS the cyanobacterium Synechococcus PCC 6301 and the bactea A Trace of Pyruvate Is Produced by Ribulose-P2 Carboxyl- rium Rhodospirillum rubrum also partitionedsimilar portion were the same, ase-The products of complete carboxylation of ribulose-P, of their productt o pyruvate. All ofthese ratios was 0.68 f 0.05 catalyzed by spinach ribulose-P2carboxylase in the presence within experimental error, and their mean elimination of the phosphate moiety in a similar manner to enediol phosphates, and thiswas confirmed in studies of the behavior of the six-carbon intermediate, 3-keto-carboxyarabinitol-P, (II), in solution. Above pH 11, 3-keto-carboxyarabinitol-P, hydrolyzes to yield a molecule each of P-glycerate, pyruvate, andPi (Lorimer etal., 1986). The lattertwo species are the products expected for the p elimination reaction of the aci-carbanion (IV) formed, along with a molecule of P glycerate, by heterolyticfission of the C2-C3 bond of the hydrated, gemdiol form of 3-keto-carboxyarabinitol-P2 (111). Here we report that pyruvateis, indeed, a significant product of ribulose-P, carboxylase's catalytic process, and we document some of the propertiesof the pyruvate-producing activity. These observations establish the intermediacy the aciof carbanion species and further emphasize the difficulties ribulose-P, carboxylase has in restraining suchhighly reactive intermediates.

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HO, /CHzOQm

H, O

,

~

5
1. SCHEME ribulose-P2carboxylase.
HC-OH

C

catalytic The

sequence of
I HC-OH I HC-OH

__
C=O H+

c;HZopo,'

-o/
HO

C

II

C-OH

II
I

coz

H&-COCi I
I

HO-&OOI HOC-OH I 1

/

1
_I

z

cooI HC-OH
CH20POT

HC-OH

HC-OH

I

I
CHzOP03'

I
CHzOP03=

I
CHzOPO3=

CH20Q'

I

I

II

Ill

CHzOPOT I HOCH I COO'

Ribulose-P2 Pyruvate Production by

Carboxylase/Oxygenase

9449

TABLE I Hatio ( ~ / p > ' r u ~ a t e produced to ribulose-I-', consumed by various ribulosc-I', carboxylases in various conditions Pyruvate production was measured spectrophotometrically as described for Fig. 2 . Below pH 7, the buffer was Pipes-NaOH; between p H 7 and 8.3, it was Hepps-NaOH or Tris-HCI; for pH 8.6, it was Hicine-NaOH; for p H 9.1, it was Ches-NaOH. The divalent metal ion concentration was 15-20 mM, except for the experiment with Mn" (and its Mi'+ control) where the concentration was 4.5 mM and 0.9 CO, mM EDTA was also present. When the calculated concentration was lower than 50 p ~ bovine erythrocyte carbonic anhydrase (0.10 , mg/ml) wasadded and O,-free conditions were maintained.The ribulose-P, concentration was 1.1 mM, except when the HCOT concentration was 1 mM or lower, in which case the ribulose-P, concent ration was adjusted to half of the HCO; Concentration. When the be a solvent was predominently 'H1O, concentrated solutionof ribulosecarboxylase, lactate dehydrogenase, and carbonic anhydrase was preactivated in 'H,O containing 20 mM Hepps-NaOH, pH 8.0, 20 mM MgClr, 10 mM NaHCO:,, and 1 mM EDTA, and then a small aliquot (1.257i of the final assay volume) was added to the 'H,O-based assay mixture to initiate catalysis. Because of lower enzyme activities in 0 E, 10 15 20 'H'O, the concentrations of ribulose-P, carboxylase, carbonic anhyL l u t l o n T n n t , , nlln drase, and lactate dehydrogenase were increased to 22 p M (catalytic FIG. 1. Chromatographic separation of the "C-labeled sites), 0.35 mg/ml, and approximately 95 units/ml, respectively. The products of the ribulose-Ps carboxylase reaction. Spinach rireactions were allowed to proceed until all of the ribulose-P, was 10 consumed and the ratio was calculated from the total extent of the bulose-P, carboxylase (0.16 mg) was preincubated for min at 25 "C in a 1-ml solution containing 60 Tris-HC1, pH 8.0, 15 mM MgCI,, mM absorbance change. Confirmation that cessation of the absorbance 10 mM NaHI'CO:, (2000 cpm/nmol), and, where indicated, 0.15 mM change corresponded to exhaustion of ribulose-Pr was obtained by of monitoring "CO, fixation in parallel assays as described for 3 or NADH, and approximately 3 units rabbit muscle lactate dehydroFig. to by confirming that the expected amount of P-glycerate was present genase. Ribulose-P2 was then added 2.2 mM and, after a further 60 min, the mixture was applied a 0.5- X 5-cm column of Bio-Rad AG to after the absorbance change had ceased. P-glycerate was measured washed by observing the decrease in absorbancet 340 nm following addition 50W-X8 cation exchange resin (100-200 mesh, H+ form) and a of an aliquot of the reaction mixture to a solution containing mM through with 2 ml of H,O. The eluate plus washings was vortexed 90 under a stream of N, to remove "CO,, neutralized to pH 6 with Hepes-NaOH, pH 7.8, 17 mM MgCI,, 150 p~ NADH, 1 mM ATP, 5 NaOH, and evaporated to dryness under a streamN, at 40 "C. The of mM phosphocreatine, and the following couplingenzymes(source residue was dissolved in 330 p l of 0.013 N H,SO,, containing 0.8 mM and approximate units/ml in parentheses): creatine kinase (rabbit pyruvate, 4 mM DL-glycerate, and 4 mM DL-lactate and aliquots (50muscle, 1): phosphoglycerate kinase (yeast, 3): glyceraldehydepbosphate dehydrogenase (rabbit muscle, 1.3); t.riosephosphate isomerase 150 p l ) were chromatographed on a 0.78- X 30-cm Bio-Rad Aminex HPX-87H column with 0.013 N H,SO, as the mobile phase at a flow (rahhit muscle, 3 ) : glycerolphosphate dehydrogenase (rabbit muscle, rate of 0.6 ml/min. The absorbance of the eluate was monitored at 1). 210 nm and fractions of 180 ul were collected for scintillation countPvruvate ing. formed Enzyme Solvent Metal pH [HCO,] (percent of sourre ribulose-PI
mM

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Mi'+

Spinach Spinach Spinach Spinach Spinach Spinach Spinach Spinach Spinach Spinach Spinach Spinach Spinach

HrO H,O H,O H,O

HZ0
HLO

HZ0 HZ0
H,O
Hz0

Mi" 10 Mi'+ Mg'+ Mi'+ Mg'+ 50 Mg" Mg'+ 10Mg'+

6.4 6.9 7.3 8.0
8.3

10
5 10
11

8.6 9.1 8.3

50

5% 0.62 0.66 0.72 0.68 0.67 0.66 0.76

H,O H,0 H20

Mg" 8.3 Mgf,+ 7.8 Mn" 7.8
8.3"

0.5 10 10
9

0.64 0.64 0.63 0.70 0.76 2.2 0.65 0.71

98.5% 'HZ0 Mg"

Khodospirillum
Synechococcus
"

Mg" 7.8 Mg'+ 7.8

46 69

I
FIG. 2. Spectrophotometric measurement of pyruvate produced duringribulose-P,carboxylation. Spinachribulose-P, 25 "C ina carboxylase (1 p~ catalytic sites) was preincubated at solution containing 80 mM Tris-HCI, pH 8.0, 17 mM MgCl,, 10 mM NaHCO:,, 50 p~ NADH, and 5 gg/ml (approximately 3 units/ml) of rabbit muscle lactate dehydrogenase. Absorbance was monitored at 340 nm. Ribulose-Py(1.1mM), P-glycerate (2 mM), and carboxypentitol-PL (40PM) were added where indicated.

Uncorrected pH meter reading.

(S.E.)%. However, when theexperiment with thespinach enzyme was conducted in the presence of 98.5% 'HHrO,the partitioning toward pyruvate increased over 3-fold to a ratio of 2.2%. The fraction of product partitioned towards pyruvate increased with temperature (Fig. 4). Over twice as much pyruvate was formed at 40 as at 10 "C. A plot of the logarithm of the percentage of pyruvate uersus the inverse of t.he absolute t.emperature was linear (Fig. 4,inset). This plot is analogous to an Arrhenius plot, but its slope yields a value (4.8 Kcal/

mol) which reflects the difference in activation energies between the pyruvate-producing and P-glycerate-producing activities. K,, (COJ of the Pyruvate-producing Activity-The effect of

9450
1000

Pyruvate Production by Ribulose-P2 CarboxylaselOxygenase
,"-", the coupled assayis intrinsically insensitive and an acceptable extent of absorbance change requires ribulose-P2 concentra. of tions in excess of 200 g ~ This limitation prevented study the effect of varying the ribulose-P, concentration, in the subsaturating range, on the rate pyruvate production. of
DISCUSSION

Time, mln

Parallel measurement of I4CO2 fixation and pyruvate production. The procedure was the same as that described for Fig. 2, except that the ribulose-P, carboxylase site concentration was 5 p ~the Trisbuffer was replaced with 90 mM Hepps-NaOH, pH8.3, , and the solutions were sparged with N,. When "CO, fixation was being measured, 10.5 mM NaH"CO,, (753 cpm/nmol) was substituted lor unlabeled bicarbonate. Otherwise the solutions for the two assays were identical. When pyruvate production was being measured, the ahsorbance was monitored a t 340 nm. When "CO, fixation was being measured, 20-/*1aliquots were removed at the times shown and added t o 0.5 ml of '20% (v/v) formic acid. After drying a t 80 "C, the acidinvolatile "C was measured by scintillation counting.
:3,

FIG.

1.4

0.0

F

x

2

-0.2

d "04

-0.6

-0.8

Pyruvate wasclearlyidentifiable among the products of carboxylation of ribulose-P2catalyzed by ribulose-P2carboxylase (Fig. 1).It was not produced when the carboxylase was specifically inhibited by carboxypentitol-P2 or when P-glycerate was substituted for ribulose-P2 (Fig. 2 ) and the timecourse of pyruvate production exactly paralleled I'CO. flxation and,by inference, P-glycerate production(Fig. 3).Therefore, production of pyruvate must be an integral part of the catalytic function of ribulose-P, carboxylase. Since the pyruvate is labeled from I4CO2 (Fig. 1) and its production shows the same K,,, (CO,) as the overall carboxylation reaction, its precursor mustbe an intermediate in the carboxylase reaction sequence formedafter the carboxylation event which produces 3-keto-carboxyarabinitol-P2. There seems little doubt, therefore, that the intermediate which decays to pyruvate is the three-carbon aci-carbanion formed following scission of the C2-C3 bond of the gemdiol form of 3-keto-carboxyarabinitolP, (Scheme 2). Apparently,the active siteisnot able to restrain this highly reactive three-carbon species exclusively tothe productive catalytic pathway.Oncein every -150 turnovers (at 25 "C) (Table I), this intermediate mustp eliminate a phosphate ion to form pyruvate. Our data thus support those showing a strong isotope effect for the attachment of the proton at C-2 of P-glycerate (Hurwitz et al., 1956; Simon et al., 1964; Fiedler et al., 1967; Saver and Knowles, 1982), in establishing the intermediacy of this aci-carbanion species. The p elimination reaction of this intermediate is analogous to that of the enediol intermediate involved in the triosephosphate isomerase reaction, where the /3 elimination prod-

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0.4

3010

20

40

I

Temperature, "C

FIG. 4. Effect of temperature on partitioning towards pyruvate. Pyruvate formationwas measured spectrophotometricallyas descrihed for Fig. 2. The solution contained, in addition to ribulosecarhoxylase, 135 mM Hepps-NaOH buffer, pH 8.3, 18 mM MgCL, 15 mM NaHCO:,, 50 pM NADH, and approximately 10 units/ml of rathit muscle lactate dehydrogenase. After preactivation for approximately 10 min, the reaction was started by adding ribulose-P, t o 0.95 mM. The stated temperature was maintained with a thermostated cuvette and measured using a calibrated thermocouple which dipped into the solution. The full extent of the absorbance change corresponding to complete consumption of the rihulose-P2 was recorded. I'-glycerate was determined on aliquots of the solution aftercomplet ion ofthe reaction as described for Table I, thus allowing calculation 0 1 the ratio of pyruvate produced to ribulose-P, consumed. The inset shows a plot of' the natural logarithm of the percentage of pyruvate ( v r s u s the inverse of the absolute temperature. Its slope yieldsa value ol 4.8 Kcal/mol for the difference inactivation energiesbetween pyruvate and P-glycerate production.

P-glycerate

I
I

(eno1)pyruvate
=%Po

0

varying CO, concentration on the rate pyruvate production of wasmeasuredusing the coupledassay as described under "Experimental Procedures." A simple hyperbolic response was observed, with a K,,, of 12.5 -+ 1.0 (S.D.) VM. This value is insignificantly different from the K,,, (COz) of the normal carboxylase reaction (Edmondson et al., 1990a). Because less than 1%of the ribulose-Pa consumedis converted to pyruvate,

"upper" P-glycerate

SCHEME The final stages of the catalytic cycle of' ribulose-P? 2. carboxylase. The following intermediates or transitionstatesare shown complexed at the catalytic site: 111, hydrated form of .?-ketocarboxyarabinitol-P,; IV, carbanion (aci-acid)form of P-glycerate; V, transition state for the final protonation.

Pyruvate Production by Ribulose-P2 CarboxylaselOxygenase

9451

uct is methyl glyoxal (Campbell et al., 1979). However, the the carboxyl termini of ,B strands to the amino termini of (Y triose-phosphate isomerase intermediate /3 eliminates much helices in thefila barrel domainof the large subunit (Andersless frequently, relative to the total rate catalytic through- son et al., 1989).) Therefore, in order for the essential inverof put, than the ribulose-P2 carboxylase aci-carbanionapparsion of configuration tobe accomplished, either some of these interactions must be broken (and presumably new interacently does. The ease with which we have been able to detect pyruvate tionsformed) or there must be considerablemovement of productioncontrasts with the failure of Jaworowski et al. these active site loops with respect to each other during this final stage the catalytic of cycle. Such complicated movements (1984) to detect it by their rapid-quench method. However, the experiment of Jaworowski et al. (1984) involved the re- might be slow and could contribute to ribulose-P2 carboxylaction of a small quantity of [1-"2P]ribulose-P2 (250 pmol) ase's slow rate of catalytic turnover.Comparison of the strucwith a larger quantity of enzyme active sites (15 nmol) for 16 ture of the carbamylated active site with P-glycerate bound ms in the presence of I4CO2with a specific radioactivity of 2 with the already determined structure carboxyarabinitolwith mCi/mmol. Detection of any pyruvate formed must rely on P, bound would shed light on thisquestion. the I4C label, because the phosphorus atom will have been Stereoelectronicconsiderations also indicatethat moveactive site during catalysis mustnecessary. be eliminated. Release of "'Pi cannot be used to measure pyruvate ment within the formation because :"Pi is also released from other acid-labile The aci-carbanion will be most resistant to /3 elimination (to intermediates earlier in the carboxylase reaction sequence. form enolpyruvate) when the bridge oxygen of the phosphate Completeconversion of the ribulose-Pn to products would is in the same plane as thedouble bond, as is represented in have resulted in the fixation of only 1110 dpm of I4C. In the Scheme 2. This is because there will be minimalorbital same experiment, itwas estimated, from the amount of acid- overlap between the C-0 bond to the phosphate and the .rr (Rose, 1981). 1, labile "'Pi released, that the reaction intermediates (I, 1 111, system of the double bond in this condition is in exand IV (if present), Scheme 1) accumulated to a collective However, 3-keto-carboxyarabinitol-P2 bound an total of 12.3% of the ribulose-P2 supplied. Even in the unlikely tended configuration and thebridge oxygen is positioned very circumstance that all of this "'Pi release was attributable to far away from the plane of the bond joining the carboxyl the aci-carbanion, it would have produced only 137 dpm of carbon to C-2 (Andersson et al., 1989), which will become the radioactivity in theform of pyruvate uponacidification, mak- double bond of theaci-carbanion.Furthermore, asimilar ing its detection (as lactate after a chromatographic proce- requirement for coplanarity also affects the stability of the dure) rather difficult. Lesser (and more reasonable) amounts five-carbon enediol intermediate earlier in the reaction seof the aci-carbanion would have escaped detection. We may quence and thebridge oxygen cannot adopt a single position conclude that the aci-carbanionis not an abundant interme- which would be coplanar with the doublebonds of both diate in the carboxylase reaction, but more sensitive rapid- intermediates.Therefore, considerablemovement mustbe atanystage quench experiments would be required to establish its level necessary to stabilize theaci-carbanionand, during that movement when the bridge oxygen is out of the precisely. Another possible unproductive reaction to which the aci- plane of the double bond, the phosphategroup will be particcarbanion might be susceptible would be its stereochemically ularly vulnerable to @ elimination (Rose, 1981). This would site. incorrect protonation to form L-P-glycerate. Indeed, there is lead to enolpyruvate formation at the catalytic an unconfirmed report that 10%of the P-glycerate produced On the other hand, if the aci-carbanion intermediatewere by ribulose-P, carboxylase is of the L isomeric form ( B r a n d h to escape fromthe active site, would @ eliminate very readily it et al., 1980).However, since complete carboxylation with in solution. This is known because alkaline hydrolysis of 3pyruvate '"C02 of a limiting amount of ribulose-P2 yields amounts of keto-carboxyarabinitol-P2yields predominantly et D-P-glycerate (measured by the enzymatic assay described in from the "upper" three carbons (Lorimer al., 1986).Perhaps the legend of Table I which presumably recognizes only the D the active site cannot afford to bind the aci-carbanion very isomer) which are consistent with the amount of I4CO2fixed tightly because of its resemblance to the product, P-glycerate. (data not shown), it seems unlikely that the extent of L-P- Since P-glycerate must be released rapidly before the next glycerate production canbe this large. Whether or not smaller cycle of catalysis can commence, an active site conformation traces of the L isomer are formed remains a question for future which bound the aci-carbanion tight enough to prevent any study. significant release might be susceptible to severe product Conversion of the gemdiol form of 3-keto-carboxyarabini- inhibition by P-glycerate. two molecules of D-P-glyCerate tol-Pn (111) (Scheme2)to The constancy of the fraction of product partitioned to requires that the stereochemical configuration about C-2 of pyruvate between pH 6.4 and 9.1 (Table I) could be interthe intermediate be inverted to form the "upper" molecule of preted to mean that the acidic group (BH in Scheme 2) which P-glycerate. This requires that C-1 and its attachedphosphate donates the proton which becomes attached to C-2 of P moiety must move a considerable distance with respect to the glycerate does not change its ionization state significantly in carboxyl and hydroxyl groups attached to C-2, first to form this pHrange. This would probably eliminate carboxyl groups the planar aci-carbanion (IV), and then the transition state and thehydroxyl of the aci-carbanion's phosphate group from (V) for proton transfer to P-glycerate. The structure of the consideration, as well as the t-amino groups of the catalyticatalytic site, as revealed by crystallographic studies of the cally essential lysines, 334 and 175. Lysine 334 of carbamyspinach ribulose-P, carboxylase with bound carboxyarabini- lated spinach ribulose-Pn carboxylase has a pK, of 9.0 and the tol-P, (Andersson et al., 1989), shows that the C-1 phosphate pK, of lysine166 of carbamylated Rhodospirillum rubrum group and the carboxyl and hydroxyl groups attached to C-2 ribulose-Pn carboxylase (analogous to lysine 175 of the spinare all firmly tethered. The phosphate interacts with main- ach enzyme) is 7.9 (Hartman et al., 1985). However, there is chain N atoms in loop 8, the carboxyl interacts with lysine a caveatthatmust be attachedtothis reasoning. These 334 in loop 6, and thecarboxyl and hydroxyl groups are both conclusions would be invalid if a proton-abstracting basic coordinated to the active site metal ion which is, in turn, groupwitha pK, similar to that of the proton donor was coordinated carbamylated to lysine 201 andother acidic instrumental in producing the aci-carbanion( e g . by abstractgroups inloop 2. (These loops are thesequences which connect ing a proton from one of the hydroxyl groups attached to C-3

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9452

Pyruvate Production

by RibuEose-P2 CarboxylaselOxygenase

of the gemdiol form of 3-keto-carboxyarabinitol-PZ initiate the reaction catalyzed by phosphoglycerate mutase, whose to cleavage of the C-2/C-3 bond). In this circumstance, both the presence in the chloroplast has been questioned (Stitt and ap production of the aci-carbanion and its protonation would be Rees, 1979). Direct incorporation of 14C02 into pyruvate also affected similarly by pH variation with little effect on the providesaready explanation for therapiditywith which steady-state level of the aci-carbanion. pyruvate and its transamination product alanine become carIt is particularly noteworthy that the ratio between pyru- boxyl-labeled from I4CO2 in the leaves of some species (Kenvate and P-glycerate production (in 'H20 at 25 "C) is also nedy and Laetsch, 1974). invariantinthe face of varying C 0 2 concentration,with Since pyruvate is not inhibitory and is readily and usefully metabolized, theremightbelittle selective advantage for differentdivalentmetalions attheactivesite,andwith ribulose-P2 carboxylases from different sources with widely ribulose-P, carboxlyase to evolve a more potent mechanism differing kinetic properties and turnover rates (Table I). All for protonatingitsaci-carbanionintermediateinorderto of these variables, in addition to pH, will affect the rate of suppress pyruvate formation totally. catalytic through-put and, therefore, the of aci-carbanion rate Acknowledgments-We are grateful to G. H. Lorimer for helpful formation. So it is clear that partitioning between pyruvate and P-glycerate as products is unaffectedby the rate of aci- discussions and toB. Entsch for comments on the manuscript. carbanion formation. One suspects that there may be some REFERENCES intrinsic mechanistic reason the singular constancy in the for S., Schneider, Lundqvist, relative rates of p elimination and protonation of the aci- Andersson, I., Knight, Lorimer, G. H.G., Lindqvist, Y., 3 7 , 229-234T., Brandkn, C.-I., and (1989) Nature 3 carbanion. p elimination is a unimolecular reaction not reAndrews, T. J., and Ballment, B. (1983) J . Biol. Chem. 2 5 8 , 7514quiring addition of other atoms from the enzyme or solvent, 7518 and this may confer on this reactionsome degree of indiffer- Andrews, T. J., and Lorimer, G. H. (1987) in The Biochemistry of Plants: A Comprehensive Treatise, Vol. 10, Photosynthesis (Hatch, ence to conditions within the active site. But the protonation reaction mustbe similarly indifferent to changes in the active M. D., and Boardman, N. K., eds) pp. 131-218, Academic Press, New York site which might be expectedwhen pH is varied or when Andrews, T. J.(1988) J. Biol. Chem. 263, 12213-12219 different metals are present or between the active sites of Brandbn, R., Nilsson, T., and Styring, S. (1980) Biochem. Biophys. different ribulose-P2 carboxylases. In particular, the reactivity Res. Commun. 92, 1297-1305 of the proton donor seems t o be remarkably constant. This Calvin, M. (1956) J. Chem. Soc. 1895-1915 might be easier to understandif the protonwas donated by a Campbell, I. D., Jones, R. B., Kiener, P. A., and Waley, S. G. (1979) Biochem. J. 179,607-621 water molecule, rather thanby an acidic group on theenzyme. J., Badger, Berry, J. A. It is possible to alter the partitioning ratio towards pyru- Collatz, G. Institution of M. R., Smith, C., and 78, 171-175 (1979) Carnegie Washington Yearbook vate, however, and we discovered twoways of doing this. First, Edmondson, D. L., Badger, M. R., and Andrews, T. J. (1990a) Plant the ratiodepended on temperature,with higher temperatures Physiol. 9 3 , 1376-1382 favoring pyruvate formation. The activation energy barrier Edmondson, D. L., Badger, M. R., and Andrews, T. J. (1990b) Plant Physiol. 9 3 , 1390-1397 for the productive protonation must be lower than that for Edmondson, D. L., Badger, M. R., and Andrews, T. J. (1990~) Plant the unproductive @ elimination. The data indicate that the Physiol. 93,1383-1389 difference in enthalpy between the transition states of the Edmondson, D. L., Kane, H. J., and Andrews, T. J. (1990d) FEBS two reactions could be as much as 4.8 Kcal/mol (Fig.4). Lett. 260, 62-66 Second, whenthe solvent was predominantly 'H20, partition- Fiedler. F.. Mullhofer. G.. Trebst, A,. and Rose. I. A. (1967) Eur. J. . . Biochem. 1,395-399 ing towards pyruvate (at 25 "C) increased more than %fold , . hydrogen isotope effect Hartman. F. C.. Milanez. S.. and Lee.E. H. (1985) J. Biol. Chem. (Table I). This is consistent with the 260,13968-13975 for the transfer of the proton to P-glycerate observed previ- Hunvitz, J., Jakoby, W. B., and Horecker, B. L. (1956) Biochim. ously (Hunvitz et al., 1956; Simon et al., 1964; Fiedler et al., Biophys. Acta 2 2 , 194-195 1967; Saver and Knowles, 1982) which shows that proton Jaworowski, A,, Hartman, F.C., and Rose, I. A. (1984) J. Biol. Chem. 259,6783-6789 transfer must be slow relative to subsequent steps. The deuteron's lower reactivity slows the protonation reaction, allow- Kennedy, R. A,, and Laetsch, W. M. (1974) Plant Physiol. 5 4 , 608611 ing theaci-carbanion more opportunity to p eliminate t o Lorimer, G. H., Andrews, T. J., Pierce, J., and Schloss, J. V. (1986) pyruvate. Phil. Trans. R. Soc. Lond. B 313, 397-407 The amount of pyruvate formed by this abortive p elimi- Morell, M. K., Kane, H. J., and Andrews, T. J. (1990) FEBS Lett. 265,41-45 nation reaction (approximately 0.7% of the ribulose-Ps carboxylated a t 25 "C (TableI)) does not represent a serious loss Paulsen, J. M., and Lane, M. D. (1966) Biochemistry 5,2350-2357 t o the energy budget of photosynthesis as a whole. (When Pierce,, J., Andrews, T. J., and Lorimer, G. H. (1986) J . Biol. Chem. 2 6 1 10248-10256 pyruvate isproduced instead of P-glycerate, the equivalentof Rose, I. A. (1981) Phil. Trans. R. Soc. Lond. B 2 9 3 , 131-143 one high-energy phosphoester bond iswasted.) Nevertheless, Saver, B. G., and Knowles, J. R. (1982) Biochemistry 2 1 , 5398-5403 it does represent a significant flux of carbon outof the Calvin Schloss, J. V., and Lorimer, G . H. (1982) J . Biol. Chem. 257, 46914694 cycle and this may contribute substantially to pyruvate-reD., Heineke, quiring processes in the chloroplast, such as fatty acid and Schulze-Siebert, 76,465-471 D., Scharf, H., and Schultz, G. (1984) Plant Physiol. amino acid synthesis (Schulze-Siebertet al., 1984). Moreover, Simon, H., Dorrer, H.-D., and Trebst,A. (1964) 2. Naturjorsch. 19b, pyruvate production by ribulose-P2 carboxylaseprovidesa 734-744 direct route to pyruvate within the chloroplast bypasses Stitt, M., and ap Rees, T. (1979) Phytochemistry 1 8 , 1905-1911 which
, I

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