Pyruvate is a by-product of catalysis by ribulosebisphosphate carboxylase/oxygenase

Pyruvate is a minor product of the reaction catalyzed by ribulosebisphosphate carboxylase/oxygenase from spinach leaves. Labeled pyruvate was detected, in addition to the major labeled product, 3-phosphoglycerate, when 14CO2 was the substrate. Pyruvate production was also measured spectrophotometrically in the presence of lactate dehydrogenase and NADH. The Km for CO2 of the pyruvate-producing activity was 12.5 microM, similar to the CO2 affinity of the 3-phosphoglycerate-producing activity. No pyruvate was detected by the coupled assay when ribulose 1,5-bisphosphate was replaced by 3-phosphoglycerate or when the carboxylase was inhibited by the reaction-intermediate analog, 2'-carboxyarabinitol 1,5-bisphosphate. Therefore, pyruvate was not being produced from 3-phosphoglycerate by contaminant enzymes. The ratio of pyruvate produced to ribulose bisphosphate consumed at 25 degrees C was 0.7%, and this ratio was not altered by varying pH or CO2 concentration or by substituting Mn2+ for Mg2+ as the catalytically essential metal. The ratio increased with increasing temperature. Ribulose-bisphosphate carboxylases from the cyanobacterium Synechococcus PCC 6301 and the bacterium Rhodospirillum rubrum also catalyzed pyruvate formation and to the same extent as the spinach enzyme. When the reaction was carried out in 2H2O, the spinach carboxylase increased the proportion of its product partitioned to pyruvate to 2.2%. These observations provide evidence that the C-2 carbanion form of 3-phosphoglycerate is an intermediate in the catalytic sequence of ribulose-bisphosphate carboxylase. Pyruvate is formed by beta elimination of a phosphate ion from a small portion of this intermediate.     

Analysis of catalytic subunit microheterogeneity in ribulosebisphosphate carboxylase/oxygenase from Nicotiana tabacum

Urea isoelectric focusing of dissociated, carboxymethylated Nicotiana tabacum ribulose-1,5-bisphosphate carboxylase/oxygenase reveals catalytic subunit microheterogeneity. Aggregated or nonaggregated sucrose gradient-purified preparations and the crystalline protein displayed essentially identical large subunit multiple polypeptide patterns. Various pretreatments which fully dissociate the holoenzyme did not alter catalytic subunit microheterogeneity. Direct comparison of the carboxymethylated and noncarboxymethylated crystalline and sucrose gradient-purified proteins demonstrated that the large subunit multiple polypeptide pattern was not an artifact of carboxymethylation. The inclusion of the seryl protease inhibitor phenylmethylsulfonyl fluoride during purification of the holoenzyme did not affect the large subunit multiplicity. However, the addition of leupeptin, a potent thiol proteinase inhibitor, to all solutions during purification of the native protein markedly reduced large subunit polypeptide L₃ and increased the staining of polypeptide L₂, suggesting that L₃ is a leupeptin-sensitive proteinase degradation product of L₂. Polypeptide L₁ also appeared to be a purification-related artifact, but derived from a modification of L₂ other than that which yielded L₃. We conclude that polypeptide L₂ is the single, native isoelectric form of the catalytic subunit of tobacco ribulosebisphosphate carboxylase/oxygenase.     

Mutations in the small subunit of ribulosebisphosphate carboxylase affect subunit binding and catalysis

Fully functional Synechococcus PCC 6301 ribulose 1,5-bisphosphate carboxylase-oxygenase (kcat = 11.8 s-1) was assembled in vitro following separate expression of the large- and small-subunit genes in different Escherichia coli cultures. The small subunits were expressed predominantly as monomers, in contrast to the large subunits which have been shown to be largely octameric when expressed separately [Andrews, T. J. (1988) J. Biol. Chem. 263, 12213-12219]. This separate expression system was applied to the study of mutations in the amino-terminal arm of the small subunit, which is one of the major sites of contact with the large subunit in the assembled hexadecamer. It enabled the effects of a mutation on the tightness of binding of the small subunit to the large-subunit octamer to be distinguished from the effects of the same mutation on catalysis carried out by the assembled complex when fully saturated with mutant small subunits. This important distinction cannot be made when both subunits are expressed together in the same cell. Substitutions of conserved amino acid residues at positions 14 (Ala, Val, Gly, or Asp instead of Thr) and 17 (Cys instead of Tyr), which make important contacts with conserved large-subunit residues, were introduced by site-directed mutagenesis. All mutant small subunits were able to bind to large subunits and form active enzymes. A potential intersubunit hydrogen bond involving the Thr-14 hydroxyl group is shown to be unimportant. However, the binding of Gly-14, Asp-14, and Cys-17 mutant small subunits was weaker, and the resultant mutant enzymes had reduced catalytic rates compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of ribulosebisphosphate carboxylase large subunit binding protein

The large subunit binding protein, an abundant plastid protein implicated in the assembly of ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO), has been highly purified from leaves of Pisum sativum. The 720 kilodaltons purified binding protein is composed of two types of subunits of 60 and 61 kilodaltons. Highly specific polyclonal antibodies have been raised against the binding protein. The antibodies do not cross-react with the large subunit nor do anti-RubisCO antibodies cross-react with the binding protein. A higher molecular weight form of the binding protein is immunoprecipitated from products of P. sativum polysomes translated in a wheat-germ system, indicating that the binding protein is synthesized by cytoplasmic ribosomes. Immunoblotting reveals the presence of binding protein in extracts of tobacco, wheat and barley leaves and castor bean endosperm.

The previously reported dissociation of the binding protein-large subunit complex upon addition of ATP in vitro has been confirmed and the fates of the dissociated subunits further investigated. The dissociated binding protein subunits are not phosphorylated or adenylated in vitro by added ATP.

     

An engineered change in substrate specificity of ribulosebisphosphate carboxylase/oxygenase

The potential for altering the specificity of ribulosebisphosphate carboxylase/oxygenase toward gaseous substrates is explored through a modest perturbation of the active site microenvironment. Specifically, replacement of active site Glu-48 with carboxy-methylcysteine is achieved in a two-step process in which the catalytically incompetent Cys-48 mutant protein is first generated and then treated with iodoacetic acid. This regimen of concerted site-directed mutagenesis and chemical modification, effectively lengthening the glutamyl side chain by insertion of a sulfur atom between the beta- and gamma-methylene groups, results in a protein possessing 4-6% of wild-type carboxylase activity. Concomitantly, the engineered enzyme exhibits a specificity factor 5-fold lower than that of wild-type enzyme. This represents the first example of a major change in substrate specificity, albeit in favor of oxygenation, effected by structural alteration of an active site side chain.     

Active site histidine in spinach ribulosebisphosphate carboxylase/oxygenase modified by diethyl pyrocarbonate

[3H] Diethyl pyrocarbonate was synthesized [Melchior, W. B., & Fahrney, D. (1970) Biochemistry 9, 251-258] from [3H] ethanol prepared by the reduction of acetaldehyde by NaB3H4. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach was inactivated with this reagent at pH 7.0 the presence of 20 mM Mg2+, and tryptic peptides that contained modified histidine residues were isolated by reverse-phase high-performance liquid chromatography. Labeling of the enzyme was conducted in the presence and absence of the competitive inhibitor sedoheptulose 1,7-bisphosphate. The amount of one peptide that was heavily labeled in the absence of this compound was reduced 10-fold in its presence. The labeled residue was histidine-298. This result, in combination with our earlier experiments [Saluja, A. K., & McFadden, B. A. (1982) Biochemistry 21, 89-95], suggests that His-298 in spinach RuBisCO is located in the active site domain and is essential to enzyme activity. This region of the primary structure is strongly conserved in seven other ribulosebisphosphate carboxylases from divergent sources.     

Synthesis of large subunit of ribulosebisphosphate carboxylase by thylakoid-bound polyribosomes from spinach chloroplasts

Intact chloroplasts were isolated from developing first leaves of spinach. The chloroplasts were broken and separated into an extensively washed membrane (thylakoid) fraction and a soluble (stroma) fraction. The membrane fraction contained polyribosomes with properties similar to those of thylakoid-bound polyribosomes of other organisms. The distribution of mRNA for large-subunit ribulosebisphosphate carboxylase (LS) was determined by translating RNA from chloroplasts, thylakoids, and stroma in a wheat germ cell-free translation system. LS translation product was identified by immunoprecipitation with antibody to LS from spinach, electrophoresis of the immunoprecipitated product, and fluorography. At least 44% of translatable chloroplast LS-mRNA was in the washed thylakoid fraction. Thylakoid-bound LS-mRNA was in polyribosomes since LS was produced by thylakoids in an Escherichia coli cell-free translation system under conditions where initiation did not take place. Our results demonstrate that membrane-bound polyribosomes can synthesize the stroma-localized polypeptide LS, and suggest that the thylakoids may be an important site of its synthesis.     

Sequences of two active-site peptides from spinach ribulosebisphosphate carboxylase/oxygenase

Two tryptic peptides from spinach ribulosebisphosphate carboxylase/oxygenase that contain the essential lysyl residues derivatized by the affinity label 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate were subjected to sequence analyses. The sequences of these peptides are -Tyr-Gly-Arg-Pro-Leu-Leu-Gly-Cys-Thr-Ile-Lys-Pro-Lys- and -Leu-Ser-Gly-Gly-Asp-His-Ile-His-Ser-Gly-Thr-Val-Val-Gly-Lys-Leu-Glu-Gly-Glu-Arg-, respectively. The reagent moiety is covalently attached to the internal lysyl residue in each peptide.     

Complete primary structure of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Of the 14 cyanogen bromide fragments derived from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase, four are too large to permit complete sequencing by direct means [F. C. Hartman, C. D. Stringer, J. Omnaas, M. I. Donnelly, and B. Fraij (1982) Arch. Biochem. Biophys. 219, 422-437]. These have now been digested with proteases, and the resultant peptides have been purified and sequenced, thereby providing the complete sequences of the original fragments. With the determination of these sequences, the total primary structure of the enzyme is provided. The polypeptide chain consists of 466 residues, 144 (31%) of which are identical to those at corresponding positions of the large subunit of spinach ribulosebisphosphate carboxylase/oxygenase. Despite the low overall homology, striking homology between the two species of enzyme is observed in those regions previously implicated at the catalytic and activator sites.     

A novel role for light in the activation of ribulosebisphosphate carboxylase/oxygenase

The effect of modifying calcium concentration on the expression of the photosynthesis circadian rhythm was examined in Euglena gracilis, Klebs strain Z. Expression of the oxygen evolution rhythm required the presence of both extracellular and intracellular calcium. Several treatments were found to uncouple the rate of the light reactions from the biological clock. In the presence of these chemical agents, the rate of oxygen evolution increased steadily throughout the light portion of the light/dark cycle, instead of showing a peak of activity in the middle of the light cycle. Oxygen evolution was uncoupled from the biological clock when extracellular calcium concentrations were altered by the presence of EGTA or LaCl₃. Uncoupling was also observed when intracellular calcium concentrations were disrupted by the use of Ca²⁺ channel blockers, the intracellular Ca²⁺ antagonist 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, or by disrupting expression of the inositol trisphosphate system. Uncoupling was also observed when the diacylglycerol signaling system, which activates kinase C, was inhibited by acridine orange. The inhibition was reversed by the presence of phorbol esters which activate the kinase. It was concluded that both the inositol trisphosphate and diacylglycerol signaling systems were required for the expression of the oxygen evolution rhythm generated by the biological clock.     

The sites for catalysis and activation of ribulosebisphosphate carboxylase share a common domain

The complexation of ribulosebiphosphate carboxylase with CO2, Mg2+, and carboxyarabinitol bisphosphate (CABP) to produce the quaternary enzyme-carbamate-Mg2+-CABP complex closely mimics the formation of the catalytically competent enzyme-carbamate-Mg2+-3-keto-CABP form during enzymatic catalysis. Quaternary complexes were prepared with various metals (Mg2+, Cd2+, Mn2+, Co2+, and Ni2+) and with specifically 13C-enriched ligands. 31P and 13C NMR studies of these complexes demonstrate that the activator CO2 site (carbamate site), the metal binding site, and the substrate binding site are contiguous. It follows that both the carboxylase and oxygenase activities of this bifunctional enzyme are influenced by the structures of the catalytic and activation sites.     

A soluble chloroplast protein catalyzes ribulosebisphosphate carboxylase/oxygenase activation in vivo

Ribulosebisphosphate carboxylase/oxygenase (EC 4.1.1.39) (rubisco) must be fully activated in order to catalyze the maximum rates of photosynthesis observed in plants. Activation of the isolated enzyme occurs spontaneously, but conditions required to observe full activation are inconsistent with those known to occur in illuminated chloroplasts. Genetic studies with a nutant of Arabidopsis thaliana incapable of activating rubisco linked two chloroplast polypeptides to the activation process in vivo. Using a reconstituted light activation system, it was possible to demonstrate the participation of a chloroplast protein in rubisco activation. These results indicate that a specific chloroplast enzyme, rubisco activase, catalyzes the activation of rubisco in vivo.     

Restoration of activity to catalytically deficient mutants of ribulosebisphosphate carboxylase/oxygenase by aminoethylation

Substitutions for active-site lysyl residues at positions 166 and 329 in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been shown to abolish catalytic activity. Treatment of the Cys-166 and Cys-329 mutant proteins with 2-bromoethylamine partially restores enzyme activity, presumably as a consequence of selective aminoethylation of the thiol group unique to each protein. Amino acid analyses, slow inactivation of the wild-type carboxylase by bromoethylamine, and the failure of bromoethylamine to restore activity to the corresponding glycyl mutant proteins support this interpretation. The observed facile, selective aminoethylations may reflect an active site microenvironment not dissimilar to that of the native enzyme. Catalytic constants of these novel carboxylases, which contain a sulfur atom in place of a specific lysyl gamma-methylene group, are significantly lower than that of the wild-type enzyme. Furthermore, the aminoethylated mutant proteins form isolable complexes with a transition state analogue, but with compromised stabilities. These detrimental effects by such a modest structural change underscore the stringent requirement for lysyl side chains at positions 166 and 329. In contrast, the aminoethylated mutant proteins exhibit carboxylase/oxygenase activity ratios and Km values that are unperturbed relative to those for the native enzyme.     

A critical arginine in the large subunit of ribulose bisphosphate carboxylase/oxygenase identified by site-directed mutagenesis

Rapid inactivation by phenylglyoxal of ribulose bisphosphate carboxylase/oxygenase (ribulose-P2 carboxylase) from the cyanobacterium Anacystis nidulans suggests the presence of an essential arginine, the modification of which is reduced in the presence of the substrate ribulose bisphosphate. Arginine 292 in the large subunit of ribulose-P2 carboxylase from A. nidulans was chosen for site-directed mutagenesis studies on the basis of the complete conservation of this residue in corresponding sequences of ribulose-P2 carboxylase from divergent organisms. Arginine 292 was changed to leucine and to lysine by directed mutagenesis using suitable plasmids and the bacteriophage M13. Both substitutions resulted in the production of purifiable holoenzyme with no activity after expression in Escherichia coli.     

Amino-acid sequence of the large subunit of d-ribulosebisphosphate carboxylase/oxygenase from Nicotiana tabacum

The complete amino acid sequence of the large subunit of the ribulosebisphosphate carboxylase (3-phospho-d-glycerate carboxy-lyase (dimirizing), ED 4.1.1. 39) from Nicotiana tabacum has been determined by alignment of tryptic, chymotryptic and cyanogen bromide fragments. The sequence is — except for positions 284 (glycine instead of cysteine) and 377 (valine instead of glutamic acid) — in agreement with the one deduced from gene sequencing (Shinozaki, K. and Sugiura, M. (1982) Gene 20, 91–102). However, the protein chemical determination yields additional information not evident from nucleotide sequencing: (1) The amino terminus is proteolytically processed and appears inhomogeneous; (2) The amino acid sequence is dimorphic in positions 394 and 405 (and possibly in position 23). The latter observation proves that for the large subunit of ribulosebisphosphate carboxylase from N. tabacum there exist at least two (if not more) slightly different genes.     

Ionization constants of two active-site lysyl epsilon-amino groups of ribulosebisphosphate carboxylase/oxygenase

Trinitrobenzene sulfonate rapidly inactivates ribulosebisphosphate carboxylase/oxygenase from both spinach and Rhodospirillum rubrum. With large molar excesses of the reagent, the reactions obey pseudo-first order kinetics and the rates of inactivations are directly proportional to the concentrations of trinitrobenzene sulfonate; thus, there is no indication of reversible complexation of reagent with enzyme. Saturating levels of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduce the rates of inactivations but do not prevent them, thereby suggesting that the groups subject to arylation remain accessible in the enzyme complexed with competitive inhibitor. Characterization of tryptic digests of the inactivated enzymes reveals that Lys-166 of the R. rubrum enzyme and Lys-334 of the spinach enzyme are the only major sites of arylation. Both of these lysines have been assigned to the catalytic site by prior affinity labeling studies and are found within highly conserved regions of primary structure. As a monoanion over a wide pH range, trinitrobenzene sulfonate, for which the carboxylase lacks high affinity, can thus be used to determine the pKa values of the two active-site lysyl epsilon-amino groups. Based on the pH dependency of inactivation of the R. rubrum enzyme by trinitrobenzene sulfonate, the epsilon-amino group of Lys-166 exhibits a pKa of 7.9 and an intrinsic reactivity (ko) of 670 M-1 min-1. In analogous experiments, Lys-334 of the spinach enzyme exhibits a pKa of 9.0 and a ko of 4500 M-1 min-1. Under deactivation conditions (i.e. in the absence of CO2 and Mg2+), the pKa of Lys-334 becomes 9.8 and the ko is increased to 26,000 M-1 min-1. By comparison, the reaction of trinitrobenzene sulfonate with N-alpha-acetyl-lysine reveals a pKa of 10.8 and a ko of 1250 M-1 min-1. The spinach carboxylase, catalytically inactive as a consequence of selective arylation of Lys-334, still exhibits tight binding of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate. Therefore, Lys-334 is not required for substrate binding and may serve a role in catalysis. The unusually low pKa of Lys-166 argues that this residue is also important to catalysis rather than substrate binding.     

Essentiality of the small subunit (B) in the catalysis of RuBP carboxylase/oxygenase is not related to substrate-binding in the large subunit (A)

The small subunit (B) of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase from Aphanothece halophytica is absolutely required for the catalysis, but depletion of subunit B does not significantly affect the formation of the quaternary complex-[enzyme.activator CO2.Mg.carboxyarabinitol bisphosphate] in the catalytic core. The inhibition of RuBP carboxylase activity by the reaction of the epsilon-amino group of a lysine in the RuBP-binding site with pyridoxal 5-P is the same whether subunit B is added to the catalytic core before or after the inactivating reaction. The function of subunit B is not related to the substrate binding.     

Function of Lys-166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase as examined by site-directed mutagenesis

Affinity labeling and comparative sequence analyses have placed Lys-166 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at the active site. The unusual nucleophilicity and acidity of the epsilon-amino group of Lys 166 (pKa = 7.9) suggest its involvement in catalysis, perhaps as the base that enolizes ribulosebisphosphate (Hartman, F.C., Milanez, S., and Lee, E.H. (1985) J. Biol. Chem. 260, 13968-13975). In attempts to clarify the role of Lys-166 of the carboxylase, we have used site-directed mutagenesis to replace this lysyl residue with glycine, alanine, serine, glutamine, arginine, cysteine, or histidine. All seven of these mutant proteins, purified by immunoaffinity chromatography, are severely deficient in carboxylase activity; the serine mutant, which is the most active, has a kcat only 0.2% that of the wild-type enzyme. Although low, the carboxylase activity displayed by some of the mutant proteins proves that Lys-166 is not required for substrate binding and argues that the detrimental effects brought about by amino acid substitutions at position 166 do not reflect gross conformational changes. As demonstrated by their ability to tightly bind a transition-state analogue (2-carboxyarabinitol 1,5-bisphosphate) in the presence of CO2 and Mg2+, some of the mutant proteins undergo the carbamylation reaction that is required for activation of the wild-type enzyme. Since Lys-166 is required neither for activation (i.e. carbamylation by CO2) nor for substrate binding, it must be essential to catalysis. When viewed within the context of previous related studies, the results of site-directed mutagenesis are entirely consistent with Lys-166 functioning as the base that initiates catalysis by abstracting the C-3 proton from ribulosebisphosphate. An alternative possibility that Lys-166 acts to stabilize a transition state in the reaction pathway cannot be rigorously excluded.     

Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue

Ribulosebisphosphate carboxylase/oxygenase is reversibly activated by the reaction of CO2 with a specific lysyl residue (Lys191 of the Rhodospirillum rubrum enzyme) to form a carbamate that coordinates an essential Mg2+ cation. Surprisingly, the Lys191----Cys mutant protein, in the presence of CO2 and Mg2+, exhibits tight binding of the reaction intermediate analogue 2-carboxyarabinitol bisphosphate [Smith, H. B., Larimer, F. W., & Hartman, F. C. (1988) Biochem. Biophys. Res. Commun. 152, 579-584], a property normally equated with effective coordination of the Mg2+ by the carbamate. Catalytic ineptness of the Cys191 mutant protein, despite its ability to coordinate Mg2+ properly, might be due to the absence of the carbamate nitrogen. To investigate this possibility, we have evaluated the ability of exogenous amines to restore catalytic activity to the mutant protein. Significantly, the Cys191 protein manifests ribulose bisphosphate dependent fixation of 14CO2 when incubated with aminomethanesulfonate but not ethanesulfonate. This novel activity reflects a Km value for ribulose bisphosphate which is not markedly perturbed relative to wild-type enzyme, a Km for Mg2+ which is in fact decreased 10-fold, and rate saturation with respect to aminomethanesulfonate (Kd = 8 mM). Chromatographic and spectrophotometric analyses reveal the product of CO2 fixation to be D-3-phosphoglycerate, while turnover of [1-3H]ribulose bisphosphate into [3H]phosphoglycolate confirms oxygenase activity. We conclude that aminomethanesulfonate restored ribulosebisphosphate carboxylase/oxygenase activities to the Cys191 mutant protein by providing a nitrogenous function which satisfies a catalytic demand normally met by the carbamate nitrogen of Lys191.