Solvation of CO2 in water: effect of RuBP on CO2 concentration in bundle sheath of C4 plants.

An understanding of the temperature-dependence of solubility of carbon dioxide (CO2) in water is important for many industrial processes. Voluminous work has been done by both quantum chemical methods and molecular dynamics (MD) simulations on the interaction between CO2 and water, but a quantitative evaluation of solubility remains elusive. In this work, we have approached the problem by considering quantum chemically calculated total energies and thermal energies, and incorporating the effects of mixing, hydrogen bonding, and phonon modes. An overall equation relating the calculated free energy and entropy of mixing with the gas-solution equilibrium constant has been derived. This equation has been iteratively solved to obtain the solubility as functions of temperature and dielectric constant. The calculated solubility versus temperature plot excellently matches the observed plot. Solubility has been shown to increase with dielectric constant, for example, by addition of electrolytes. We have also found that at the experimentally reported concentration of enzyme RuBP in bundle sheath cells of chloroplast in C4 green plants, the concentration of CO2 can effectively increase by as much as a factor of 7.1-38.5. This stands in agreement with the observed effective rise in concentration by as much as 10 times.

Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the fixation of atmospheric CO(2) in photosynthesis, but tends to form inactive complexes with its substrate ribulose 1,5-bisphosphate (RuBP). In plants, Rubisco is reactivated by the AAA(+) (ATPases associated with various cellular activities) protein Rubisco activase (Rca), but no such protein is known for the Rubisco of red algae. Here we identify the protein CbbX as an activase of red-type Rubisco. The 3.0-Å crystal structure of unassembled CbbX from Rhodobacter sphaeroides revealed an AAA(+) protein architecture. Electron microscopy and biochemical analysis showed that ATP and RuBP must bind to convert CbbX into functionally active, hexameric rings. The CbbX ATPase is strongly stimulated by RuBP and Rubisco. Mutational analysis suggests that CbbX functions by transiently pulling the carboxy-terminal peptide of the Rubisco large subunit into the hexamer pore, resulting in the release of the inhibitory RuBP. Understanding Rubisco activation may facilitate efforts to improve CO(2) uptake and biomass production by photosynthetic organisms.

Residues that influence in vivo and in vitro CbbR function in Rhodobacter sphaeroides and identification of a specific region critical for co-inducer recognition.

CbbR is a LysR-type transcriptional regulator (LTTR) that is required to activate transcription of the cbb operons, responsible for CO2 fixation, in Rhodobacter sphaeroides. LTTR proteins often require a co-inducer to regulate transcription. Previous studies suggested that ribulose 1,5-bisphosphate (RuBP) is a positive effector for CbbR function in this organism. In the current study, RuBP was found to increase the electrophoretic mobility of the CbbR/cbb(I) promoter complex. To define and analyse the co-inducer recognition region of CbbR, constitutively active mutant CbbR proteins were isolated. Under growth conditions that normally maintain transcriptionally inactive cbb operons, the mutant CbbR proteins activated transcription. Fourteen of the constitutively active mutants resulted from a single amino acid substitution. One mutant was derived from amino acid substitutions at two separate residues that appeared to act synergistically. Different mutant proteins showed both sensitivity and insensitivity to RuBP and residues that conferred constitutive transcriptional activity could be highlighted on a three-dimensional model, with several residues unique to CbbR shown to be at locations critical to LTTR function. Many of the constitutive residues clustered in or near two specific loops in the LTTR tertiary structure, corresponding to a proposed site of co-inducer binding.

Conformational flexibility of the regulatory site of phosphoribulokinase as demonstrated with bifunctional reagents.

Phosphoribulokinase (PRK) is one of several chloroplastic enzymes whose activity is regulated by thiol-disulfide exchange via thioredoxin. Activation entails reduction of an active-site disulfide bond between Cys16 and Cys55. Bifunctional cross-linking reagents have been used to approximate the interresidue distance between Cys16 and Cys55, an issue which impinges on the relative conformational states of the activated and deactivated forms of the enzyme. Spinach PRK is rapidly inactivated by stoichiometric levels of 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone (FNPS) or 1,5-difluoro-2,4-dinitrobenzene (DFNB), which span 9 and 3.5 A, respectively. ATP, but not ribulose 5-phosphate, retards the rate of inactivation, suggesting that modification has occurred at the nucleotide binding domain of the active site. Sulfhydryl modification is indicated by partial reversibility of inactivation as effected by exogenous thiols. Tryptic mapping by reverse-phase chromatography of [14C]carboxymethylated enzyme, subsequent to its reaction with either FNPS or DFNB, demonstrates modification of Cys16 and Cys55 by both reagents, and formation of only one major chromophoric peptide in each case. On the basis of the sequence analysis of the purified chromophoric peptides, Cys16 and Cys55 are cross-linked by both FNPS and DFNB. Thus, the intrasubunit distance between the beta-sulfhydryls of Cys16 and Cys55 is dynamic rather than static. Diminished conformational flexibility upon oxidation of the regulatory sulfhydryls to a disulfide may be partially responsible for the concomitant loss of enzymatic activity.

Activation of in situ glycolytic flux by bisphosphorylated compounds: studies in porous rat adipocytes.

By carefully permeabilizing eukaryotic cells such that intracellular enzymes are largely retained, an opportunity is created to explore the regulation of in situ flux. This is particularly important since the latter may not be accurately represented by kinetic measurements of isolated, solubilized enzymes from disrupted cells. In this study the action of fructose 2,6-diphosphate (F2,6DP) and other bisphosphorylated sugars which purportedly activate phosphofructokinase-1 (PFK-1; EC 2.7.1.11) were studied. Using porous adipocytcs and initiating flux with radiolabeled glucose 6-phosphate, the regulation of lactate production under both 0.1 and 1.0 mM ATP conditions by F2,6DP, glucose 1,6-diphosphate (G1,6DP), ribulose 1,5-diphosphate (R1,5DP), 2,3 diphosphoglycerate (2,3DPG), and mannose 6-phosphate (M6P) was examined. Studied at 1, 5, and 25 microM concentrations, F2,6DP and 2,3DPG significantly (and to the same extent) augmented glycolysis compared to control (at 0.1 mM ATP, the respective glycolytic rates--as % above control--at these three above-mentioned concentrations for F2,6DP were 60, 84, and 77%, whereas for 2,3DPG they were 84, 105, and 179%; at 1 mM ATP, the F2,6DP effect was 88, 99, and 121%, and for 2,3DPG it was 52, 89, and 96%). Stimulation by these compounds was less obvious at higher glycolytic flux rates (saturating amounts of G6P). Amongst this group, and only at 1.0 mM ATP, the sole other positive effector was 25 microM R1,5DP. The measured fat cell content of G1,6DP was 24 +/- 4 microM (n = 3); at this concentration no significant effect on glycolysis was observed. Examining the effects of 2,3DPG (25 microM) on proximal glycolysis (to triose phosphates) revealed there was a modest, but significant, 41% increase over basal; in contrast, under the exact same conditions, F2,6DP caused a 123% increase. Separate experiments also examined the effect of F2,6DP, 2,3DPG, and G1,6DP on glycolysis at 5 and 25 microM in the presence of a physiologic cytosolic ATP/ADP ratio and free cation concentrations. Under these conditions, F2,6DP and 2,3DPG remained pre-eminent in their stimulatory prowess, inducing 27-71% increases over control, while G1,6DP remained ineffectual. These studies support a locus of action of 2,3DPG on overall glycolysis which is distal to the triose phosphates. M6P was ineffective at all concentrations. In conclusion, F2,6DP is the pre-eminent in situ regulator of in situ adipocyte glycolysis, especially at higher ATP levels, although other sugars containing two phosphoryl groups may under certain conditions cause activation.

Some kinetic properties of pyruvate kinase from Trypanosoma brucei.

We have studied the kinetics of the allosteric interactions of pyruvate kinase from Trypanosoma brucei. The kinetics for phosphoenolpyruvate depended strongly on the nature of the bivalent metal ions. Pyruvate kinase activated by Mg2+ had the highest catalytic activity, but also the highest S0.5 for phosphoenolpyruvate, while the opposite was true for pyruvate kinase activated by Mn2+. The reaction rates of Mg(2+)-pyruvate kinase and Mn(2+)-pyruvate kinase were clearly allosteric with respect to phosphoenolpyruvate, while the kinetics with Co(2+)-pyruvate kinase were hyperbolic. However, Co(2+)-pyruvate kinase was still sensitive to heterotropic activation. Trypanosomal pyruvate kinase is unique in that the best activator was fructose 2,6-bisphosphate. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate were also strong heterotropic activators, which were much more effective than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. In the presence of the heterotropic activators, the sigmoidal kinetics with respect to phosphoenolpyruvate and the bivalent metal ions were modified as were the concentrations of phosphoenolpyruvate and the bivalent metal ions needed to attain the maximal activity. Maximal activities were not significantly changed with Mg2+ and Mn2+ as the activating metal ions. Moreover, with Co2+ and fructose 2,6-bisphosphate or ribulose 1,5-bisphosphate or 5-phosphorylribose 1-pyrophosphate, the maximal activity was significantly reduced. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate resembled fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate in their action in that the K0.5 values for the former 3 compounds increased when Mg2+ was replaced by Co2+, while the K0.5 for fructose 1,6-bisphosphate and glucose 1,6-bisphosphate increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Ribulose bisphosphate-induced, slow conformational changes of spinach ribulose bisphosphate carboxylase cause the two types of inflections in the course of its carboxylase reaction.

The cause of the inflection in the course of the carboxylase reaction and the changes in the functioning form of spinach ribulose bisphosphate carboxylase (RuBisCO) during the reaction were elucidated by relating the activity to the protein conformation of RuBisCO using a fluorescence probe, 2-p-toluidinylnaphthalene sulfonate. The activity of RuBisCO in the linear phase was 50 to 60% of that in the initial burst at 0.5 to 1.0 mM ribulose bisphosphate (RuBP) and 65 to 80% at 2 to 5 mM RuBP. The amount and the progress of the decrease in the activity during the reaction had a close relationship to a change in the protein conformation of RuBisCO. The enzyme, the substrate binding sites of which were masked beforehand with carboxyarabinitol bisphosphate, still showed a change of its protein conformation upon addition of RuBP, suggesting that RuBisCO has two (substrate and regulatory) RuBP-binding sites per RuBisCO promoter. RuBisCO required over 2 mM RuBP for binding on the regulatory sites. Both sites also bound 6-phosphogluconate. When both sites were masked with 6-phosphogluconate beforehand, the course of the subsequent carboxylase reaction was linear with time. From these results, I propose that the inflection in the course of the reaction of spinach RuBisCO is a hysteretic response of the enzyme to RuBP bound to both substrate and regulatory sites.

Determination of effector molecules in L-arabinose-induced bulge formation and lysis of Escherichia coli IFO 3545.

L-Ribulose 5-phosphate (L-Ru5P) was identified as the primary effector molecule of L-arabinose-induced bulge formation in Escherichia coli IFO 3545 observed in nutrient broth with 5% (w/v) sodium chloride. Hyperinduction of L-arabinose isomerase was due to exogenous sodium chloride and the resulting alteration in the balance of the L-arabinose-metabolizing enzymes resulted in accumulation of L-Ru5P. L-Ru5P induced the lysis of an L-arabinose-negative, L-Ru5P 4-epimerase-less mutant, ara-207, even when directly added to the medium but was not active against the wild-type strain. Some L-arabinose-utilizing (L-arabinose-resistant) revertants of ara-207 were still sensitive to L-Ru5P, indicating the involvement of another mutation in L-Ru5P-sensitivity other than genetic lack of L-Ru5P 4-epimerase. Among the various pentose phosphate esters tested, only L-Ru5P could induce lysis of ara-207. The lytic activity of L-Ru5P was attributed to its effect on bacterial sugar nucleotide metabolism which caused secondary accumulation of uridine 5'-diphosphate galactose (UDPGal), which provoked lysis induction.

Affinity labeling of spinach phosphoribulokinase subsequent to S-methylation at Cys16.

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme, S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparent Kd of 3-4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [14C]reagent per mole of enzyme subunit. Amino acid analysis of the [14C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.

Evidence for different kinases in thylakoid protein phosphorylation.

Thylakoid protein phosphorylation was facilitated in darkness by using the ferredoxin-NADPH system. CoCl2 and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) were potent inhibitors of LHCP (light-harvesting chlorophyll-binding protein) phosphorylation, but 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea and atrazine had no significant effect. Differential effects on phosphorylation of the 9 kDa polypeptide and LHCP were observed in darkness with DBMIB and certain other inhibitors specific for Photosystem-II electron transport. Similarly, during illumination of intact chloroplasts or of the reconstituted chloroplast system, a differential action of bicarbonate was observed on the relative phosphorylation of the two proteins. The degree of phosphorylation of the 9 kDa polypeptide was increased in the presence of bicarbonate compared with its absence, whereas that of LHCP was relatively unchanged. Changes in the degree of phosphorylation of the 32 kDa polypeptide in these experiments did not correlate consistently with changes in phosphorylation of either LHCP or the 9 kDa polypeptide, although changes in the 32 kDa polypeptide more often paralleled phosphorylation of the 9 kDa polypeptide rather than the phosphorylation of LHCP. These observations suggest that the protein kinase that phosphorylates LHCP is distinct from that which phosphorylates the 9 kDa polypeptide.

Inhibition of ribulose bisphosphate carboxylase by substrate ribulose 1,5-bisphosphate.

Substrate ribulose bisphosphate is a potent and a weak inhibitor of the rate of CO2/Mg2+ activation in the carboxylase purified from spinach leaves and Rhodospirillum rubrum, respectively. At 2 degrees C, the concentration of ribulose bisphosphate required for 50% inhibition of the initial rate of CO2/Mg2+ activation was less than 0.4 microM for the spinach enzyme, but between 67 and 270 microM for the R. rubrum carboxylase. Activator 14CO2 trapping experiments demonstrated that ribulose bisphosphate inhibits activation by excluding activator CO2 from the spinach enzyme. The reason for the different sensitivities to inhibition by substrate was evident from equilibrium binding studies with the inactive enzyme forms which indicated that the KD (ribulose bisphosphate) was 0.021 microM for spinach enzyme and 5.9 microM for the R. rubrum protein. Inhibition of activation, however, was not explained by the equilibrium binding results alone. Ribulose bisphosphate was observed to dissociate very slowly from the inactive spinach enzyme (at 2 degrees C, kOFF = 4.9 X 10(-5) s-1). The release of substrate from the inactive R. rubrum carboxylase was much more rapid, with a minimum value for kOFF estimated at 5 X 10(-3) s-1 at 2 degrees C. We conclude that strong inhibition of CO2/Mg2+ activation in the spinach enzyme is mediated by the tight binding and slow release of ribulose bisphosphate, which prevent activator CO2 and Mg2+ from binding to the protein. Weak inhibition of activation in the R. rubrum enzyme results from a larger KD value and a more rapid exchange of ribulose bisphosphate, which allow activator CO2 and Mg2+ to bind to the free enzyme between successive substrate-binding events.

Competition of pyridoxal 5'-phosphate with ribulose 1,5-bisphosphate and effector sugar phosphates at the reaction centers of the spinach ribulose 1,5-bisphosphate carboxylase/oxygenase.

The stimulation of the carboxylase reaction by effectors of ribulose 1,5-bisphosphate carboxylase/oxygenase displays higher sensitivity towards pyridoxal 5'-pyridoxal 5'-phosphate inhibition than the catalytical process itself. Pyridoxal 5'-phosphate binding to the enzyme is not affected by the modulators 6-phosphogluconate and fructose 1,6-bisphosphate at low concentrations at which these agents stimulate the carboxylation rate. At higher concentrations these sugar phosphates protect the enzyme against pyridoxal 5'-phosphate inhibition in a similar fashion like the substrate ribulose 1,5-bisphosphate. Such protection experiments in combination with spectrophotometrical studies of pyridoxal 5'-phosphate binding demonstrate two binding states of ribulose 1,5-bisphosphate at the reaction centers of the enzyme with different requirements for Mg2+. 6-Phosphogluconate functions as protector only in the presence of Mg2+. Our results imply a competition between pyridoxal 5'-phosphate and substrate or effector sugar phosphates at the reaction centers of the spinach carboxylase. It is proposed that the pyridoxal 5'-phosphate inhibition of the stimulatory activity of these effectors originates from a modification of the regulatory sites of the enzyme caused by pyridoxal 5'-phosphate binding to the catalytical sites.

The use of 1-anilino-8-naphthalene sulfonate as fluorescent probe for conformational studies on ribulose-1,5-bisphosphate carboxylase.

The influence of Mg2+ ions and temperature on the structure of the enzyme ribulose-1,5-bisphosphate carboxylase was investigated using the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS). The binding of ANS to the enzyme molecule caused a significant increase of fluorescence emission which was further enhanced by the addition of Mg2+. The temperature dependence of the fluorescence emission indicated a conformational change of the enzyme between 12 and 24 degrees C. The Mg2+ and the temperature effects were additive. ANS itself did not change the conformation of the enzyme. The influence of the substrates carbon dioxide and ribulose-1,5-bisphosphate, and the effect of the pH of the medium and of a sulfhydryl reducing reagent on fluorescence emission were analysed.

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios.

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) FROM SPINACH CHLOROPLASTS IS STRONGLY REGULATED BY THE RATIO OF NADPH/NADP+, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP+ ratio. With a ratio of NADPH/NADP+ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited. This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP+ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions. Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP+ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP+. It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP+ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolic unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP+ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.