The Activation State of Ribulose 1,5-bisphosphate Carboxylase in Maize Leaves in Dark and Light

A spectrophotometric procedure for assay of initial and totalactivity of ribulose 1,5-bisphosphate carboxylase in maize leaveswas established. The extraction of the crude enzyme from maizeleaf tissue, which was prefrozen in liquid nitrogen, desaltingof the extract, and assay of the enzyme was completed within3 min. From experiments adding deactivated ribulose 1,5-bisphosphatecarboxylase to the leaf tissue prior to extraction it was estimatedthat the maximum extent of activation during extraction, desaltingand assay was 8%. In predarkened leaves the enzyme showed 67to 84% of maximal activation while in preilluminated leavesthe enzyme showed 89 to 98% of maximal activation. These resultsindicate that deactivation of the enzyme in the dark is nota reason for the previous finding of a transient peak of ribulose1,5-bisphosphate in maize leaves during induction of photosynthesis[Usuda (1985) Plant Physiol. 78: 859–864]. This transientincrease in the substrate level upon illumination might be explainedby the presence of an unknown negative effector for ribulose1,5-bisphosphate carboxylase in vivo in leaf tissue in the dark,or limiting CO₂ supply to the enzyme during the induction period. (Received May 30, 1985; Accepted August 16, 1985)     

Ribulose 1,5-bisphosphate carboxylase from the thermophilic, acidophilic alga, Cyanidium caldarium (Geitler). Purification, characterisation and thermostability of the enzyme.

An an initial stage in the study of proteins from thermophilic algae, the enzyme ribulose 1,5-bisphosphate carboxylase 2-phospho-D-glycerate carboxylyase (dimerizing, EC 4.1.1.39) was purified 11-fold from the thermophilic alga Cyandium caldarium, with a 24% recovery. This purified enzyme appeared homogeneous on polyacrylamide gels and could be dissociated into two subunit types of molecular weights 55,000 and 14,900. The optimal assay temperature was 42.5 degrees C, whilst enzyme purified from Chlorella spp. showed maximum activity at 35 degrees C. The thermostability of Cyanidium ribulose 1,5-bisphosphate carboxylase was considerably greater than that of the Chlorella enzyme, and the presence of Mg2+ and HCO-3 further enhanced this heat stability. A break in the Arrhenius plot occured at 20 degrees C for Chlorella ribulose 1,5-bisphosphate carboxylase and 36 degrees C for the enzyme from Cyanidium. It is suggested that the thermostability of Cyanidium ribulose 1,5-bisphosphate carboxylase is a result of an inherent stability of the enzyme molecule which permits efficient CO2 fixation at high temperatures but results in low activity in the mesophilic temperature range.     

Identification of the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase as a Substrate for Transglutaminase in Medicago sativa L. (Alfalfa)

Extracts prepared from floral meristematic tissue of alfalfa (Medicago sativa L.) were investigated for expression of the enzyme transglutaminase in order to identify the major protein substrate for transglutaminase-directed modifications among plant proteins. The large polymorphic subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase in alfalfa, with molecular weights of 52,700 and 57,600, are major substrates for transglutaminase in these extracts. This was established by: (a) covalent conjugation of monodansylcadaverine to the large subunit followed by fluorescent detection in SDS-polyacrylamide gels; (b) covalent conjugation of [¹⁴C]putrescine to the large subunit with detection by autoradiography; (c) covalent conjugation of monodansylcadaverine to the large subunit and demonstration of immunocross-reactivity on nitrocellulose transblot of the modified large subunit with antibody prepared in rabbits against dansylated-ovalbumin; (d) demonstration of a direct dependence of the rate of transglutaminase-mediated, [¹⁴C]putrescine incorporation upon the concentration of ribulose, 1,5-bisphosphate carboxylase/oxygenase from alfalfa or spinach; and (e) presumptive evidence from size exclusion chromatography that transglutaminase may cofractionate with native molecules of ribulose 1,5-bisphosphate carboxylase/oxygenase in crude extracts. Analysis of the primary structure of plant large subunit has revealed numerous potential glutaminyl and lysyl sites for transglutaminase-directed modifications of ribulose 1,5-bisphosphate carboxylase/oxygenase.     

Rate-limiting factors in leaf photosynthesis. I. Carbon fluxes in the calvin cycle

Rates of photosynthesis of spinach leaves were varied by varying light intensity and CO₂ concentration. Metabolism of the leaves was then arrested by freezing them in liquid nitrogen. Chloroplasts were isolated by a nonaqueous procedure. In the chloroplast fractions, levels of intermediates of the carbon reduction cycle were determined and considered in relation to the photosynthetic flux situation of the leaves at the time before freezing. During induction of photosynthesis, ribulose 1,5-bisphosphate levels increased in parallel with CO₂ fixation. In the steady state, a similar relation between ribulose 1,5-bisphosphate levels and CO₂ uptake was observed at light intensities between 0 and 50 W·m⁻². A further increase in light intensity increased CO₂ fixation rates but not ribulose 1,5-bisphosphate levels. Increasing the CO₂ concentration resulted in increased CO₂ uptake, whereas ribulose 1,5-bisphosphate levels decreased. Even under CO₂ saturation, ribulose 1,5-bisphosphate levels were about 100 nmol/mg chlorophyll corresponding to about 3.5 mM ribulose 1,5-bisphosphate in the chloroplast stroma. This suggests that even under CO₂ saturation, ribulose-1,5-bisphosphate carboxylase limits photosynhetic CO₂ uptake. Mass action ratios calculated from measured metabolite levels demonstrated that the thermodynamic gradient required for the regeneration of ribulose 1,5-bisphosphate from hexosephosphate and triosephosphate increased considerably as photosynthetic flux increased. Similar calculations revealed that the enzymatic apparatus responsible for the reduction of 3-phosphoglycerate to dihydroxyacetone phosphate is not displaced much from equilibrium even under maximum rates of photosynthesis at saturating CO₂. The same is true for aldolase. Fructose-1,6-bisphosphatase also did not limit Calvin cycle turnover. Only at very low light intensities and during the first minutes of the induction period was the ratio of fructose 1,6-bisphosphate to fructose 6-phosphate high. This observation was more readily explained in terms of fructose 1,6-bisphosphate binding to ribulose-1,5-bisphosphate carboxylase than by a rate limitation imposed by insufficient activation of fructose-1,6-bisphosphatase.     

Intermediates formed by the Co2+-activated ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach studied by electron paramagnetic resonance spectroscopy

Two enzyme-metal-bound intermediates formed by the Co2+-activated ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) have been studied by electron paramagnetic resonance (EPR) spectroscopy. Their rates of approach to a stationary state are different and their relative amounts at steady state are dependent on the concentration of ribulose 1,5-bisphosphate. It is therefore proposed that enzyme-metal-coordinated ribulose 1,5-bisphosphate and an enzyme-metal-coordinated enediolate anion of it, where bound ribulose 1,5-bisphosphate appears first, constitute the two EPR-detectable intermediates, respectively.     

Retention of the oxygens at C-2 and C-3 of D-ribulose 1,5-bisphosphate in the reaction catalyzed by ribulose-1,5-bisphosphate carboxylase.

Ribulose-1,5-bisphosphate carboxylase catalyzes the conversion of D ribulose 1,5-bisphosphate and CO2 to 3-phospho-D-glycerate, with retention of the oxygen atoms at both C-2 and C-3 of the substrate. This observation is consistent with mechanistic pathways involving an enediol intermediate and eliminates suggested mechanisms that involve covalent intermediates between the enzyme and ribulose 1,5-bisphosphate in which the substrate oxygen at C-2 or C-3 is compulsorily lost.     

Ribulose-1,5-bisphosphate carboxylase: enzyme-catalyzed appearance of solvent tritium at carbon 3 of ribulose 1,5-bisphosphate reisolated after partial reaction

When ribulose 1,5-bisphosphate is allowed to react with carbon dioxide in tritiated water in the carboxylation reaction catalyzed by ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum, the ribulose 1,5-bisphosphate reisolated after partial reaction is found to be labeled. The specific radioactivity of the remaining substrate pool rises during the course of the reaction. Experiments in deuterium oxide show that the isotopic label resides on carbon 3. Earlier failures to detect this exchange process probably derive from the use of enzyme that was, in the absence of carbon dioxide, inactive. The present results provide direct evidence for the intermediacy of the enediol between C-2 and C-3 of ribulose 1,5-bisphosphate and show that the enolization step is at least partially rate limiting in the overall carboxylase reaction. The specific radioactivity of the product 3-phospho-D-glycerate remains constant throughout the course of the reaction at about one-sixth that of the solvent. This strengthens the argument against the involvement of "sticky" protons in the reaction.     

Interactions between ribulose-1,5-bisphosphate carboxylase and stromal metabolites. II. Corroboration of the role of this enzyme as a metabolite buffer

The capacity of ribulose-1,5-bisphosphate carboxylase to bind reversibly chloroplast metabolites which are the substrates for both thylakoid and stromal enzymes was assessed using spinach chloroplasts and chloroplast extracts and with pure wheat ribulose-1,5-bisphosphate carboxylase. Measurements of the rate of coupled electron flow to methyl viologen in ‘leaky’ chloroplasts (which retained the chloroplast envelope and stromal enzymes but which were permeable to metabolites) and also with broken chloroplasts and washed thylakoids were used to study the effects of binding ADP and inorganic phopshate to ribulose-1,5-bisphosphate carboxylase. The presence of ribulose-1,5-bisphosphate carboxylase significantly altered the values obtained for apparent K_m for inorganic phosphate and ADP of coupled electron transport. The K_m (P_i) in washed thylakoids was 60–80 μM, in ‘leaky’ chloroplasts it was increased to 180–200 μM, while in ‘leaky’ chloroplasts preincubated with KCN and ribulose 1,5-bisphosphate the value was decreased to 40–50 μM. Similarly, the K_m (ADP) of coupled electron transport in washed thylakoids was 60–70 μM, in ‘leaky’ chloroplasts it was 130–150 μM and with ‘leaky’ chloroplasts incubated in the presence of KCN and ribulose 1,5-bisphosphate a value of 45–50 μM was obtained. The ability of ribulose 1,5-bisphosphate carboxylase to reduce the levels of free glycerate 3-phosphate in the absence of ribulose 1,5-bisphosphate was examined using a chloroplast extract system by varying the concentrations of stromal protein or purified ribulose 1,5-bisphosphate carboxylase. The effect of binding glycerate 3-phosphate to ribulose-1,5-bisphosphate carboxylase on glycerate 3-phosphate reduction was to reduce both the rate an the amount of NADPH oxidation for a given amount of glycerate 3-phosphate added. The addition of ribulose 1,5-bisphosphate reinitiated NADPH oxidation but ATP or NADPH did not. Incubation of purified ribulose-1,5-bisphosphate carboxylase with carboxyarabinitolbisphosphate completely inhibited the catalytic activity of the enzyme and decreased inhibition of glycerate-3-phosphate reduction. Two binding sites with different affinities for glycerate 3-phosphate were observed with pure ribulose-1,5-bisphosphate carboxylase.     

Rapid kinetics of an N-terminal mutant of cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase

The transient changes in absorption of visible light upon addition of ribulose 1,5-bisphosphate to Co2(+)-activated ribulose-1,5-bisphosphate carboxylase/oxygenase were used to show altered catalytic properties of a mutant form of the enzyme from Anacystis nidulans. The mutant form of the enzyme had a modified N-terminus and a 10-fold greater Km for ribulose 1,5-bisphosphate than the natural cyanobacterial enzyme.     

Regulation of photosynthesis in nitrogen deficient wheat seedlings

Nitrogen effects on the regulation of photosynthesis in wheat (Triticum aestivum L., cv Remia) seedlings were examined. Ribulose 1,5-bisphosphate carboxylase/oxygenase was rapidly extracted and tested for initial activity and for activity after incubation in presence of CO₂ and Mg²⁺. Freeze clamped leaf segments were extracted for determinations of foliar steady state levels of ribulose 1,5-bisphosphate, triose phosphate, 3-phosphoglycerate, ATP, and ADP. Nitrogen deficient leaves showed increased ATP/ADP and triose phosphate/3-phosphoglycerate ratios suggesting increased assimilatory power. Ribulose 1,5-bisphosphate levels were decreased due to reduced pentose phosphate reductive cycle activity. Nevertheless, photosynthesis appeared to be limited by ribulose 1,5-bisphosphate carboxylase/oxygenase, independent of nitrogen nutrition. Its degree of activation was increased in nitrogen deficient plants and provided for maximum photosynthesis at decreased enzyme protein levels. It is suggested that ribulose 1,5-bisphosphate carboxylase/oxygenase activity is regulated according to the amount of assimilatory power.     

Inhibition and inactivation of liver aldolase

Substrate analogs xylulose 1,5-bisphosphate, glucitol 1,6-bisphosphate, α-2,5-anhydroglucitol 1,6-bisphosphate, α-, β-methyl fructofuranoside 1,6-bisphosphate, ribulose 1,5-bisphosphate, ribulose 5-phosphate, and ribose 5-phosphate and inactivating agents 1-chloro-2, 4-dinitrobenzene, 4-hydroxymercuribenzoate, and pyridoxal phosphate were examined for their effects on liver aldolase. These studies support the use of the β-anomer and acyclic form as substrate. They also suggest that the liver enzyme active site is similar to the muscle enzyme but with a much weaker 6-phosphate binding site.     

Derepression of the synthesis of D-ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.

The synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase in Rhodospirillum rubrum was greatly influenced by the conditions of culture. When grown photolithotrophically in an atmosphere containing low levels of CO2 (1.5 to 2%), enzyme synthesis was derepressed, with the result that the enzyme comprised up to 50% of the soluble protein of the cells as determined by immunological quantitation. This response was not observed when R. rubrum was grown photolithotrophically in an atmosphere of 5% CO2 in hydrogen. Similarly, the derepression of ribulose 1,5-bisphosphate carboxylase/oxygenase was observed in photoheterotrophically (butyrate)-grown cultures only after the HCO3- supply was nearly exhausted. The increase in enzyme activity observed in derepressed cultures was not paralleled by an increase in the in vivo CO2 fixation rate. Apparently, R. rubrum derepresses the synthesis of ribulose 1,5-bisphosphate carboxylase/oxygenase when exposed to low CO2 concentrations to scavenge the limited CO2 available to such cultures.     

Catalytic properties of recombinant octameric, hexadecameric, and heterologous cyanobacterial/bacterial ribulose- 1,5-bisphosphate carboxylase/oxygenase

The recent isolation of a catalytically competent recombinant octameric core of the hexadecameric ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans (Synechococcus) (B. Lee and F. R. Tabita, 1990, Biochemistry 29, 9352-9357) has provided a useful system for examining the properties of this enzyme in the absence of small subunits. Unlike most sources of hexadecameric ribulose bisphosphate carboxylase, the nonactivated Anacystis holoenzyme was not inhibited markedly by preincubation with ribulose 1,5-bisphosphate. This was also true for the Anacystis octameric core and a heterologous recombinant enzyme that comprised large subunits from Anacystis and small subunits from the bacterium Alcaligenes eutrophus, suggesting that substrate-mediated inactivation is not influenced by small subunits. In addition, the CO2/O2 specificity factor was not affected by the source of the small subunits incorporated into the structure of the hexadecameric protein, in agreement with previous in vitro heterologous reconstitution studies. The activated octameric Anacystis enzyme, however, was significantly more sensitive to inhibition by the phosphorylated effector 6-phosphogluconate than were the hexadecameric Alcaligenes and Anacystis enzymes and the heterologous Anacystis-Alcaligenes hybrid.     

Immunological determination of phosphoenolpyruvate carboxylase and the large and small subunits of ribulose 1,5-bisphosphate carboxylase in leaves of the c(4) plant pearl millet

The light-dependent development of the photosynthetic apparatus in the first leaf of the C₄ plant pearl millet (Pennisetum americanum) was monitored by immunologically determining the concentration of phospho-enolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase. A competitive enzyme-linked immunosorbent assay procedure using antibodies to the monomeric subunit of phosphoenolpyruvate carboxylase and the large and small subunit of ribulose 1,5-bisphosphate carboxylase was used to quantitate the amounts of these polypeptides in the first leaf of etiolated seedlings and etiolated seedlings exposed to light for varying periods of time. Phosphoenolpyruvate carboxylase was present in etiolated tissue; however, light stimulated its synthesis nearly 23-fold. Maximum accumulation of phosphoenolpyruvate carboxylase occurred approximately 4 days after etiolated plants were placed in the light. Both the large subunit and the small subunit of ribulose 1,5-bisphosphate carboxylase were present in leaves of etiolated seedlings. Light also stimulated the synthesis of both of these polypeptides, but at different rates. In etiolated leaves there was approximately a 3-fold molar excess of the small subunit to large subunit. Exposure of the etiolated leaves to light resulted in the molar ratio of the large subunit to the small subunit increasing to approximately 0.72. These data indicate that the net synthesis of these two polypeptides is not coordinately regulated at all times.     

Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase.

Several mutants of Rhodopseudomonas sphaeroides defective in the derepression of the enzyme ribulose 1,5-bisphosphate carboxylase have been isolated by using the unstable Tn5 vectors pJB4JI and pRK340. Transpositional insertion mutants obtained with pJB4JI were demonstrated to be incapable of increasing ribulose 1,5-bisphosphate carboxylase/oxygenase levels when grown on butyrate-bicarbonate medium or under conditions of carbon starvation, whereas the wild-type strain increased activity four- to eightfold. When the wild-type strain was starved for carbon in the presence of chloramphenicol, no derepression was observed. Crude extracts from mutant and wild-type strains had distinct and consistent differences in protein content as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chromatographic evidence indicated that mutants were defective in the regulation of only one of the two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase synthesized by R. sphaeroides.     

Dynamic, ligand-dependent conformational change triggers reaction of ribose-1,5-bisphosphate isomerase from Thermococcus kodakarensis KOD1

Ribose-1,5-bisphosphate isomerase (R15Pi) is a novel enzyme recently identified as a member of an AMP metabolic pathway in archaea. The enzyme converts d-ribose 1,5-bisphosphate into ribulose 1,5-bisphosphate, providing the substrate for archaeal ribulose-1,5-bisphosphate carboxylase/oxygenases. We here report the crystal structures of R15Pi from Thermococcus kodakarensis KOD1 (Tk-R15Pi) with and without its substrate or product. Tk-R15Pi is a hexameric enzyme formed by the trimerization of dimer units. Biochemical analyses show that Tk-R15Pi only accepts the α-anomer of d-ribose 1,5-bisphosphate and that Cys(133) and Asp(202) residues are essential for ribulose 1,5-bisphosphate production. Comparison of the determined structures reveals that the unliganded and product-binding structures are in an open form, whereas the substrate-binding structure adopts a closed form, indicating domain movement upon substrate binding. The conformational change to the closed form optimizes active site configuration and also isolates the active site from the solvent, which may allow deprotonation of Cys(133) and protonation of Asp(202) to occur. The structural features of the substrate-binding form and biochemical evidence lead us to propose that the isomerase reaction proceeds via a cis-phosphoenolate intermediate.     

Facile assay of enzymes unique to the Calvin cycle in intact cells, with special reference to ribulose 1,5-bisphosphate carboxylase.

A procedure for the facile measurement, in intact cells, of two enzymes unique to the Calvin cycle, ribulose 1,5-bisphosphate carboxylase and phosphoribulokinase, is described. The procedure involved a simple toluene treatment to render phototrophic cells permeable to the necessary substrates, effectors, and cofactors. Whole-cell ribulose 1,5-bisphosphate carboxylase activity quantitatively approximates the activity obtained in cell-free extracts. In addition, the activity measured with toluene-treated whole cells results in a stoichiometric carboxylation of ribulose 1,5-bisphosphate to phosphoglyceric acid. The assay procedures described are most convenient for determining enzyme levels as a function of growth. Moreover, such an assay should open the way to further studies on the regulation of CO₂ assimilation by direct measurement of the enzymes concerned within the cell.     

Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis

The epsilon-amino group of Lys-166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase was postulated as the essential base which initiates catalysis by abstracting the proton at C-3 of ribulose 1,5-bisphosphate (Hartman, F. C., Soper, T. S., Niyogi, S. K., Mural, R. J., Foote, R. S., Mitra, S., Lee, E. H., Machanoff, R., and Larimer, F. W. (1987) J. Biol. Chem. 262, 3496-3501). To scrutinize this possibility, the site-directed Gly-166 mutant, totally devoid of ribulosebisphosphate carboxylase activity, was examined for its ability to catalyze each of three partial reactions. When carbamylated at Lys-191 (i.e. activated with CO2 and Mg2+), wild-type enzyme catalyzed the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon reaction intermediate of the carboxylase reaction (Pierce, J., Andrews, T. J., and Lorimer, G. H. (1986a) J. Biol. Chem. 261, 10248-10256). Likewise, when carbamylated at Lys-191, the Gly-166 mutant also catalyzed the hydrolysis of this reaction intermediate. The carbamylated wild type catalyzed the enolization of ribulose 1,5-bisphosphate as indicated by the transfer of 3H radioactivity from [3-3H]ribulose, 1,5-bisphosphate to the medium. However, even when carbamylated at Lys-191, the mutant protein did not catalyze the enolization of ribulose 1,5-bisphosphate. Additionally, unlike the decarbamylated wild-type enzyme, which catalyzed the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate in the absence of Mg2+, the mutant protein was inactive in this partial reaction. These properties exclude the epsilon-amino group of Lys-166 as an obligatory participant in the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate. In contrast, these properties are consistent with the epsilon-amino group of Lys-166 functioning as an acid-base catalyst in the enolization of ribulose 1,5-bisphosphate (when the enzyme is carbamylated) and in the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate (when the enzyme is decarbamylated). Alternatively, Lys-166 may stabilize the transition states of these two partial reactions.     

Glutamine synthetase in ribulose 1,5-bisphosphate carboxylase/oxygenase deficient tobacco mutants in cell suspension culture

In two tobacco mutants lacking ribulose, 1,5-bisphosphate carboxylase/oxygenase the amount of glutamine synthetase and its activity were determined and compared with the wild type green cells. It was shown that in these two mutants glutamine synthetase protein content was six times lower than in the wild type. This situation was comparable to that found in etiolated cells where ribulose 1,5-bisphosphate carboxylase/oxygenase was absent. These observations suggest that a common regulatory mechanism might control the dual light dependent biosynthesis of both enzymes. The results have also implications concerning the efficiency of the reassimilation of ammonia by chloroplastic glutamine synthetase during the photorespiratory process.     

The orientation of substrate and reaction intermediates in the active site of ribulose-1,5-bisphosphate carboxylase

There are four possible orientations of the substrate ribulose 1,5-bisphosphate in the active site of ribulose-1,5-bisphosphate carboxylase. Distinction between these four possible orientations has been made on the basis of 31P NMR and borohydride-trapping experiments. The orientation of the reaction-intermediate analog, 2'-carboxy-D-arabinitol 1,5-bisphosphate with respect to the divalent metal ion was determined by 31P NMR studies of the quaternary complex, enzyme.CO2.Ni2+.2'-carboxyarabinitol 1,5-bisphosphate. Assignment of the phosphorus resonances of this complex was made by labeling the phosphoryl group at either C-1 or C-5 with 17O. The phosphorus atom closer to the paramagnetic metal ion, Ni2+, to which the broader of the phosphorus resonances is attributed, has been identified as that attached to C-1. When bound to the active site of carbaminated enzyme, D-ribulose 1,5-bisphosphate was reduced by sodium borohydride with absolute stereospecificity to D-arabinitol 1,5-bisphosphate. The reduction of the enzyme-bound substrate thus occurred on the Si face of the C-2 carbonyl group. These two results together establish that ribulose 1,5-bisphosphate is oriented within the active site so that 1) the phosphoryl group at C-1 is closer to the divalent metal ion than that at C-5 and 2) the Si face of the carbonyl group points to the "outside world."