934
Biochemistry 1980, 19, 934-942
Berliner, L. J., & Wong, S. S. (1975) Biochemistry 14, 4977-4982. Bevington, P. R. (1 969) Data Reduction and Error Analysis for the Physical Sciences, p 212, McGraw-Hill, New York. Clymer, D. J., Geren, C. R., & Ebner, K. E. (1976) Biochemistry 15, 1093-1097. Davies, W. L. (1936) The Chemistry of Milk, p 225, Van Nostrand, New York. Dwek, R. A. (1973) Nuclear Magnetic Resonance in Biochemistry, pp 282-283, Clarendon Press, Oxford. Farrar, T. C., & Becker, E. D. (1971) Pulse and Fourier Transform N M R , pp 20-22, Academic Press, New York. Fitzgerald, D. K., Colvin, B., Mawal, R., & Ebner, K. E. (1 970) Anal. Biochem. 36, 43-61, Geren, C. R., Magee, S. C., & Ebner, K. E. (1975) Biochemistry 14, 1461-1463. Geren, C. R., Magee, S. C., & Ebner, K. E. (1976) Arch. Biochem. Biophys. 172, 149-155. Hill, R. L., Barker, R., Olsen, K. W., Shaper, J. H., & Trayer, I. P. (1972) in Metabolic Interconcersion of Enzymes (Wieland, 0..Ed.) pp 331-346, Springer-Verlag, Berlin. Kitchen, B. J., & Andrews. P. (1974) Biochem. J . 143, 587-590. Lee, C. M., & Sarma, R . M. (1976) Biochemistry 15, 697-704.
Magee, S. C., & Ebner, K. E. (1974) J . Biol. Chem. 249, 6992-6998. Magee, S . C., Mawal, R., & Ebner, K. E. (1974) Biochemistry 13, 99-102. Mawal, R., Morrison, J . F., & Ebner, K. E. ( 1 97 1) J . Biol. Chem. 246, 7106-7iO9. Morrison, J . F., & Ebner, K. E. (1971) J . Biol. Chem. 246, 3977-3984. Nunez, H. A., & Barker, R. (1976) Biochemistry 15, 3843-3847. Powell, J. T., & Brew, K. (1974) Eur. J . Biochem. 48, 217-228. Powell, J. T., & Brew, K. (1976) J . Biol. Chem. 251, 3645-3652. Rudolph, F. B., & Fromm, H. J . (1971) J . Biol. Chem. 246, 661 1-6619. Swift, T. J., & Connick, R. E. (1962) J . Chem. Phys. 37, 307-320. Sykes, B. D., Schmidt, P. G., & Stark, G. R. (1970) J . Biol. Chem. 245, 1180-1 189. Taylor, J. S. (1969) Ph.D. Dissertation, University of Pennsyivania, No. m i 6 , 221, p Zi, University Microfiims, Ann Arbor, MI. Trayer. I . P.. & Hill, R. L. (1971) J . Biol. Chem. 246, 6666-6675.
Interaction of Ribulosebisphosphate Carboxylase/Oxygenase with Transition-State Analogues’ John Pierce,* N . E. Tolbert, and Robert Barker*
ABSTRACT: 2-C-Carboxy-~-ribitol1,5-bisphosphate and 2-Ccarboxy-D-arabinitol 1,5-bisphosphate have been synthesized, purified, and characterized. In the presence of Mg2+, 2-Ccarboxy-D-arabinitol 1,5-bisphosphate binds to ribulose-l,5bisphosphate carboxylase/oxygenase by a two-step mechanism. The first, rapid step is similar to the binding of ribulose 1,5bisphosphate or its structural analogues. The second step is a slower process ( k = 0.04 SC’) and accounts for the tighter binding of 2-C-carboxy-~-arabinitol1,5-bisphosphate (& 5 IO-” M ) than of 2-C-carboxy-~-ribitoI1,5-bisphosphate (& = 1.5 X 10” M). Both carboxypentitol bisphosphates exhibit competitive inhibition with respect to ribulose 1,5-bisphosphate. 2-C-(Hydroxymethyl)-~-ribitol 1,5-bisphosphate and 2-C-
An
essential reaction for co2fixation in all photosynthetic organisms is catalyzed by ribulose-P2 carboxylase/oxygenase.’ Interest in this enzyme has been stimulated by the discovery of the oxygenase reaction (Ogren & Bowes, 1971; Andrews et al., 1973) and its role in the glycolate pathway of photo-
’
From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824. Received September 17, 1979. Supported in part by grants from the National Institute of General Medical Sciences ( G M 21731) and the National Science Foundation (PCM 7815891). This paper is Michigan Agricultural Experiment Station Journal No. 9168. *Present address. Division of Biological Sciences, Cornell University, Ithaca, NY 14853.
0006-2960/80/0419-0934$01 .OO/O
(hydroxymethy1)-D-arabinitol 1,5-bisphosphate were also synthesized; both are competitive inhibitors with respect to ribulose 1,5-bisphosphate with Ki = 8.0 X M and Ki= 5.0 X M, respectively. Thus, the carboxyl group of 2C-carboxy-D-arabinitol 1,5-bisphosphate is necessary for maximal interaction with the enzyme. Additionally, Mg2+ is essential for the tight binding of 2-C-carboxy-~-arabinitol 1,5-bisphosphate. A model for catalysis of ribulose l,5-bisphosphate carboxylation is discussed which includes a functional role for Mg2+ in the stabilization of the intermediate 2-C-carboxy-3-keto-~-arabinitol 1,5-bisphosphate. Mechanistic implications that arise from the stereochemistry of this intermediate are also discussed.
respiration (Tolbert & Ryan, 1976). An outline for the chemical mechanism of the carboxylation reaction was predicted by Calvin (1954) even before the discovery of the enzyme (Scheme I). The initial step is the enolization of ribulose-P, (1) to form 2, which is attacked by C 0 2 to form a 2-C-carboxy-3-ketopentitol bisphosphate, 3. Addition of HzO across the bond at C-2 and C-3 of 3 yields two molecules of D-glycerate-3-P (Fiedler et al., 1967; Cooper I Abbreviations used: ribulose-P,, D-erythro-pentulose 1,5-bisphosphate; P, phosphate; P,, 1,s-bisphosphate; 13CN M R , ”C nuclear magnetic resonance spectroscopy; carboxypentitol-P2, an unresolved mixture of 2-C-(phosphohydroxymethyl)-~-ribonic acid 5-phosphate and 2-C-(phosphohydroxymethyl)-~-arabinonicacid 5-phosphate.
Q 1980 American Chemical Society
R I B U L O S E B I S P H O S P H A T EC A R B O X Y L A S E / O X Y G E N A S E
c so
935
Chart I
Scheme I C HzO PO; I
V O L . 19, N O . 5 , 1 9 8 0
-
-
-Hi
H-L-oH I H -C -OH I CHz0 PO=,
/I C-OH
-
+
~-64)~ I CHzO PO;
!I
coz
CH20P03'-
I HO-C-C02
-C-OH
-
I
I H-C-OH
F'O
I
H-LIH I
CH20P02-
I 02C-C-OH
-
CH20P03'-
I
HO-C-CHzOH
I
-OH
I
H-C-OH
H-C-OH
CHzOPO;
LH20P03'-
-L
carboyarabinitol-P,
CH,OPO?-
carboxyribitol-P,
ny droxy meth y i-
arabinitol-P,
5. CHzOPG
CHzOPO=, I
HO-h-CO; I 5.0 H-~GH I
CHzO PO;
I
____, + HzO
HO -C -H 1 -
coz H-C-OH t
CO; I H-C-OH
+ Ht
I
CHzOPG
et al., 1969; Miillhofer & Rose, 1965; Pierce et al., 1980; Weissbach et al., 1956; Jakoby et al., 1956). In addition, the oxygen atoms at C-2 and C-3 of ribulose-P2 are retained in t h e products of t h e carboxylation reaction, d i n g out the intermediacy of eneamine or dithioacetal derivatives in the reaction (Sue & Knowles, 1978; Lorimer, 1978). An analogous mechanism for the oxygenase reaction accounts for all known facts regarding the formation of ~-glycerate-3-Pand glycolate-2-P from ribulose-P, and molecular oxygen (Lorimer et al., 1973). Attempts have been made to synthesize the P-keto acid intermediate 3 (Siegel & Lane, 1973), and quenching from the steady state of the cai'bGXj'lti=itXC:iGn gives a compound with the expected properties of 3 (Sjijdin & Vestermark, 1973). The finding that a stable analogue of 3, carboxypentitol-P2, is a competitive inhibitor with respect to ribulose-P2 has been considered proof for the existence of intermediate 3 in the reaction (Wishnick et al., 1970; Siegel & Lane, 1972, 1973). These studies utilized a mixture of carboxyribitol-P2 and carboxyarabinitol-P2 that resulted from cyanide addition to ribulose-Pz. Although the studies were performed prior to 1976 when the requirement for preincubation with Mg2+ and C 0 2 for maximal enzyme activity was clearly established (Lorimer et al., 1976; Badger & Lorimer, 1976), carboxypentitol-P2was shown to require Mgz+ for maximal inhibition. The enzyme-Mg2+-carboxypentitol-P, complex was reported to have Kd < M (Siegel & Lane, 1972). These results were suggested to imply a role for Mg2+ in the stabilization of intermediate 3. In view of our interest irr the structure ofthe active site o f ribulose-P2 carboxylase/oxygenasc a n d in the USE of t h e carboxypentitol-P2 compounds by a number of workers to probe the active site of the enzyme (Miziorko & Mildvan, 1974; Ryan & Tolbert, 1975; Schloss et al., 1978; Miziorko, 1979), it was desirable to further characterize these compounds and their interaction with the enzyme. Consequently, structural analogues (see Chart I) of the reaction intermediate 3 in the carboxylation of ribulose-P, were synthesized and their interactions with ribulose-P2 carboxylase/oxygenase were examined. Materials and Methods Compounds a n d Enzymes. Sodium [I4C]bicarbonate (NaHI4CO3) was from Amersham/Searle, and potassium [14C]cyanide (KI4CN) was from New England Nuclear.
hydroxymethylribitol-P,
3,4dideoxyaldehydopentitol-P,
aldeh ydopenbtol-P,
Potassium [13C]cyanide (KI3CN) was supplied by the LOS Alamos Scientific Laboratory, University of California, LOS Alamos, NM-. Other chemicals were reagent grade or better and were used without further purification. Biochemicals were purchased from Sigma Chemical Co. Ribulose-P, carboxylase/oxygenase (EC 4.1.1.39) was purified from spinach (Ryan & Tolbert, 1975); the enzyme was stored as a precipitate in 50% saturated (NH4),S04 at 4 "C. Ribulose-P, was prepared enzymatically from D-ribose-5-P (Horecker et al., 1956). Sugar phosphates with 13Cenrichment at various carbon atoms were prepared as described previously (Serianni et ai., i979j. i,5-Dihydroxypentan-2-one-Pz was prepared by the procedure of Hartman & Barker (1965). Instrumentation. 13C N M R spectra were obtained at 15.08 MHz with a Bruker WP-60 Fourier-transform spectrometer equipped with quadrature detection. Spectra were obtained with 4000 spectral points. The spectrometer was locked to the resonance of [2H]H20in a capillary insert. Chemical shifts are given relative to external tetramethylsilane and are accurate to within 0.1 ppm. Mass spectra were obtained from a combined gas chromatograph-mass spectrometer utilizing an LKB spectrometer equipped with a computer-assisted data acquisition system. Compounds were chromatographed at 150 " C on a 2.0-m, small bore column containing 3% OV- 17. Radioactivity measurements were obtained either on a Packard Tri-Carb scintillation counter or on a LKB LS-100 scintillation counter using a toluene-Triton X- 100 mixture. Carboxyribitol-Pz [2-C-(Phosphohydroxymethyl)-~arabinonic Acid 5-PI and Carboxyambinitol-P2 [2-C-(Pbsphohydr0xymethyl)-D-ribonic Acid 5 - P ] . A 50-100 mM solution of ribulose-P, (with or without I3C and/or 14C enrichment) at pH 8.5 was added to a 0.5 M solution of KCN (with or without I3C and/or I4C enrichment) SO that the final ratio of cyanide to ribulose-P, was 1.1. The resulting nitriles were allowed to hydrolyze to the acid salts at 22 " C for 48 h. The solution was treated with excess Dowex 50(H+), filtered, concentrated to dryness in vacuo at 30 "C, and desiccated in vacuo (50.1 mmHg) at room temperature over MgC104 for 24 h. The lactones were dissolved in water and quickly adjusted to pH 5.5 with 1 M NaOH. For small amounts of material ( 5 1 mmol of total P), the solution was added to a 42 X 2 cm Dowex 1(C1-) (8% cross-linked; 200-400 mesh) column and eluted with a 4-L, linear gradient of 0.0-0.4
936
BIOCHEMlSTRY
M LiCl in 3 m M HCI a t a rate of 0.5-0.8 mL/min. For larger amounts of material, the solution was added to a 49 x 3.3 cm Dowex i(CI-) column and eluted with a 6-L, linear gradient of 0.0-0.4 M LiCl in 3 mM HCI at a rate of 0.6-1 .O mL/min. Fractions (15 mL) were collected and assayed for total phosphate (LeLoir & Cardini, 1957) or radioactivity. Peak fractions were pooled, neutralized to pH 8.0 with 1 M LiOH, and concentrated in vacuo to approximately 100 mL. The addition of a threefold molar excess of barium acetate. followed by the addition of ethanol to a final concentration of 50% (v/v), precipitated the bisphosphates as their barium salts. After at least 1 h at -20 "C, the precipitate was collected by centrifugation and twice washed with 95% ethanol.. T i e products were dissolved in water bq the addition of excess Dowex 50(HC),filtered, adjusted to pH 6.5 with 1 M NaOH, and stored at -20 "C until use. Recovery of radioactivity was usually about 90% of that applied to the Dowex I (Cl-~)column. The compounds were estimated to be at least 95% pure by "C N M R , phosphate, and gas chromatographic analyses. Prior to use with ribulose-P, carboxylase/oxygenase, the compounds were incubated at p H 9.0 for 24 h at room temperature to ensure that no lactone forms were present. Hydroxymethylribitol-P, [ 2 - C - ( H y d r o x y m e t h y l ) - ~ ribitol-PJ and Hydroxymethylarabinitol-P,[2-C-(Hydroxymerhyl)-~-arabinitol-P,]. The y-lactones of [2'-''C]carboxy.ribiiol-P22 and. [2'-13C].~r~haxyarabinitol-Pz were p r e p r e & hum- t k i r salts by Dowex 50(Hf) treatment and desiccation as described above. A 20-mL aliquot of a 0.5 M solution of N a B H 4 in 0.4 M N a 2 C 0 3was added to 50 pmol of the appropriate lactone. The reduction was terminated after 24 h a t room temperature by the addition of 2 mL of glacial acetic acid, Dowex-50(H:+) treatment, evaporation to dryness in vacuo, and repeated concentration from anhydrous methanol removed excess borate. The products were purified by column chromatography on Dowex 1(CI-) as described above. The hydroxymethyl derivatives eluted a t approximately 0.1 M LiC1, followed by the carboxy derivatives; products were collected as their barium salts, converted to the sodium salts, and stored a t -20 "C as described for the carboxypentitol bisphosphates. [2'-I3C] Hydroxymethylribitol-P, and [2'-I3C]hydroxymethylarabinitol-P2were characterized by I3C N M R analysis. The I3CNMR chemical shifts of the enriched carbons were 62.8 ppm for the ribo derivative and 63.5 ppm for thearakino derivative. The purity of these compounds was estimated to be at least 95% by I3C N M R . Aldehydopentitol-P2 [Equimolar Mixture of'2-C-(Phosphohydroxymethyl)-~-ribose-5-Pa n d 2-C-(Phosphohydroxymethyl)-~-arabinose-5-P]. An equimolar solution of KI3CN (containing K14CN) and ribulose-P, was kept a t plH 7.5 and 4 "C for 45 min. The resulting cyanohydrins were reduced on palladium-barium sulfate ( 5 % ) (Serianni et al.. 1979). Analysis by I3C N M R revealed the presence of glycosylamine derivatives after the reduction. The mixture ofglycosylamines was converted to the aldoses by incubation a t p H 8.4 for 1 2 h at room temperature. Aldehydopentitol-P, was purified on a 5 I X 2.2 cm Dowex I (formate) column by using a 4-L, linear sodium formate gradient (0.2-1.3 M, pH 6.2). The aldoses eluted at approximately 0.5 M sodium
* The branched-chain compounds used in this report are named as derivatives of the D-pentitol-P2 compounds and numbered so as to stress their structural relationship to ribulose-P,. The tertiary carbon is designated C-2. The carbon derived from cyanide (Le,, carboxyl. hydroxymethyl, or aldehydo carbon) is designated C-2'
PIERCE. TOLBERT. AND BARKER
formate. After treatment of the pooled fractions with excess Dowex 50(H+), formic acid was removed by continuous ether extraction for 24 h a t 4 "C, and the compounds were stored as their sodium salts a t -20 "C until use. Analysis by "C N M R revealed that aldehydopentitol-P, exists in solution as the cyclic, anomeric furanoses (chemical shifts: 102.9 ppm, 43%; 102.2 ppm, 8%; 98.3 ppm, 49%) with no detectable (
