Molecular evolution of ribulose-1, 5-bisphosphate carboxylase

The structural homology of the two constituent subunits (A andB) of ribulose- 1,5-bisphosphate carboxylase from various originswas determined using the statistical method of Marchalonis andWeltman [Comp. Biochem. Biophys. 38B, 609–625 (1971)].It was found that the large catalytic subunit (A) is structurallyhomologous among the enzymes of divergent origins, from primitivephotosynthetic bacteria (Bacteriophyta) through the green algae(Chlorophyta) to higher plants (Tracheophyta). In contrast,the small regulatory subunit (B) was found to be structurallyquite different among the different species. The genetic conservationof subunit A during the phylogenetic evolution of the ribulose-1,5-bisphosphatecarboxylase molecule indicates its origin from a common ancestralgene. ¹ This is paper XXXIII in the series "Structure and Functionof Chloroplast Proteins". (Received July 23, 1975; )     

Crystal structure of activated ribulose-1,5-bisphosphate carboxylase complexed with its substrate, ribulose-1,5-bisphosphate

The three-dimensional structure of the complex of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum, CO2, Mg2+, and ribulose bisphosphate has been determined with x-ray crystallographic methods to 2.6-A resolution. Ribulose-1,5-bisphosphate binds across the active site with the two phosphate groups in the two phosphate binding sites of the beta/alpha barrel. The oxygen atoms of the carbamate and the side chain of Asp-193 provide the protein ligands to the bound Mg2+ ion. The C2 and the C3 or C4 oxygen atoms of the substrate are also within the first coordination sphere of the metal ion. At the present resolution of the electron density maps, two slightly different conformations of the substrate, with the C3 hydroxyl group "cis" or "trans" to the C2 oxygen, can be built into the observed electron density. The two different conformations suggest two different mechanisms of proton abstraction in the first step of catalysis, the enolization of the ribulose 1,5-bisphosphate. Two loop regions, which are disordered in the crystals of the nonactivated enzyme, could be built into their respective electron density. A comparison with the structure of the quaternary complex of the spinach enzyme shows that despite the different conformations of loop 6, the positions of the Mg2+ ion, and most atoms of the substrate are very similar when superimposed on each other. There are, however, some significant differences at the active site, especially in the metal coordination sphere.     

Small subunit contacts in ribulose-1,5-bisphosphate carboxylase

The arrangement of subunits of ribulosebisphosphate carboxylase in solution has been studied by exposing the enzyme to the cross-linking agents tetranitromethane, dimethyl suberimidate, and dimethyl adipimidate, and the cleavable cross-linking agent, methyl 4-mercaptobutyrimidate followed by gel electrophoresis in the presence of dodecyl sulfate. All these agents caused the formation of dimers of the enzyme's small subunit, independently of protein concentration. In addition, trimers and tetramers of small subunit were detected in the mercaptobutyrimidate-treated enzyme. The data show that small subunits are closely paired in the native enzyme and may be in layers of four, or a ring of eight.     

Intermediates in the ribulose-1,5-bisphosphate carboxylase reaction

At least two intermediates of the D-ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) reaction were liberated in detectable amounts when the functioning enzyme from Rhodospirillum rubrum was quenched in acid. Using substrate labeled with 32P in C-1, [32P]orthophosphate (Pi) was found when the quenched solution was rapidly processed for extraction of Pi as the acid molybdate complex. Reaction with sodium borohydride under mildly alkaline conditions immediately after acid quenching of the carboxylase reaction decreased the amount of 32Pi that was observed by 68%. The compound whose degradation to Pi was prevented by reaction with sodium borohydride decomposed under both acid and neutral conditions with a half-time of about 5 min at 25 degrees C and was assigned to the beta-keto acid recently demonstrated for the spinach enzyme ( Schloss , J.V., and Lorimer , G.H. (1982) J. Biol. Chem. 257, 4691-4694). It was sufficiently stable upon neutralization to react productively with fresh enzyme. As substrate CO2 concentration was decreased below the steady state Km value, the proportion of the 32P that did not react with sodium borohydride increased, indicative of a second unstable intermediate that precedes the carboxylation step. The decomposition of the latter intermediate to Pi, which occurs with a t1/2 less than or equal to 6 ms, was prevented if I2 was present in the acid quench medium. These are properties expected of the 2,3- enediol form of ribulose bisphosphate. Both intermediates reach their maximum levels when product formation is most rapid and disappear when product formation is complete as expected of reaction intermediates.     

Dissociation of ribulose-1,5-bisphosphate bound to ribulose-1,5-bisphosphate carboxylase/oxygenase and its enhancement by ribulose-1,5-bisphosphate carboxylase/oxygenase activase-mediated hydrolysis of ATP

Ribulose bisphosphate (RuBP), a substrate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is an inhibitor of Rubisco activation by carbamylation if bound to the inactive, noncarbamylated form of the enzyme. The effect of Rubisco activase on the dissociation kinetics of RuBP bound to this form of the enzyme was examined and characterized with the use of ³H-labeled RuBP and proteins purified from spinach (Spinacia oleracea L.) In the absence of Rubisco activase and in the presence of a large excess of unlabeled RuBP, the dissociation rate of bound [1-³H]RuBP was much faster after a short (30 second) incubation than after an extended incubation (1 hour). After 1 hour of incubation, the dissociation rate constant (K_off) of the bound RuBP was 4.8 × 10⁻⁴ per second, equal to a half-time of about 35 minutes, whereas the rate after only 30 seconds was too fast to be accurately measured. This time-dependent change in the dissociation rate was reflected in the subsequent activation kinetics of Rubisco in the presence of RuBP, CO₂, and Mg²⁺, and in both the absence or presence of Rubisco activase. However, the activation of Rubisco also proceeded relatively rapidly without Rubisco activase if the RuBP level decreased below the estimated catalytic site concentration. High pH (pH 8.5) and the presence of Mg²⁺ in the medium also enhanced the dissociation of the bound RuBP from Rubisco in the presence of RuBP. In the presence of Rubisco activase, Mg²⁺, ATP (but not the nonhydrolyzable analog, adenosine-5′-O-[3-thiotriphosphate]), excess RuBP, and an ATP-regenerating system, the dissociation of [1-³H]RuBP from Rubisco was increased in proportion to the amount of Rubisco activase added. This result indicates that Rubisco activase-mediated hydrolysis of ATP is required for promotion of the enhanced dissociation of the bound RuBP from Rubisco. Furthermore, product analysis by ion-exchange chromatography demonstrated that the release of the bound RuBP, in an unchanged form, was considerably faster than the observed increase in Rubisco activity. Thus, RuBP dissociation was experimentally separated from activation and precedes the subsequent formation of active, carbamylated Rubisco during activation of Rubisco by Rubisco activase.     

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K _m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.     

Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by ribulose-1,5-bisphosphate epimerization and degradation products

Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P₂) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P₂, is also present. Both compounds simulate the substrate inhibition of ribulose-P₂ carboxylase/oxygenase previously reported for ribulose-P₂. Freshly prepared ribulose-P₂ had little inhibitory activity. The instability of ribulose-P₂ may be one reason for a high level of ribulose-P₂ carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P₂. Because the K_D of the enzyme/substrate complex is ≤1 μM, all ribulose-P₂ generated in situ may be stored as this complex to prevent decomposition.     

Development of ribulose-1,5-diphosphate carboxylase in castor bean cotyledons

Light was not essential for the development of ribulose-1,5-diphosphate carboxylase protein or catalytic activity in the photosynthetic cotyledons of germinating castor beans (Ricinus communis). Cotyledons developing in the dark showed higher activity than those in the light. Returning cotyledons developing in the light to darkness resulted in a significant increase in ribulose-1,5-diphosphate carboxylase activity compared to cotyledons in continuous light.     

Purification and characterization of maize ribulose-1,5-bisphosphate carboxylase

The ribulose-1,5-bisphosphate carboxylase/oxygenase purified from maize (a C₄ monocot) to homogeneity has a MW of532 000 and sedimentation coeffici     

Biosynthesis of ribulose-1,5-bisphosphate carboxylase in spinach leaf protoplasts

Spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts sustain protein-synthesizing activity as measured by the incorporation of [¹⁴C]-leucine into the protein fraction both in the light and in the dark. By the immunoprecipitation of ribulose-1,5-bisphosphate (RuP₂) carboxylase with rabbit antibody raised against the purified spinach enzyme preparation, it was found that approximately 7% of the total radiocarbon incorporated into the protein fraction in the light was in the carboxylase molecules. However, there was no measurable net increase observed in the content of the enzyme protein in the experimental conditions employed. It was found that both chloramphenicol and cycloheximide inhibited the incorporation of [¹⁴C]leucine into RuP₂ carboxylase and its constituent subunits, as measured by the immunoprecipitation of the enzyme molecule and its subunits, A and B.     

Free subunits of ribulose-1,5-bisphosphate carboxylase in pea leaves

Pea leaves, supplied with [³⁵S]methionine, were homogenized and a crude hypotonic soluble fraction was centrifuged on sucrose gradients to separate fully assembled ribulose-1,5-biphosphate (RuBP) carboxylase from any free or partially assembled carboxylase subunits. Slowly sedimenting subunits of the enzyme were identified in upper fractions of the sucrose gradient, using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), isoelectric focussing, and immune precipitation. The presence of these subunits in low molecular weight form was shown not to be due to artefactual dissociation of the enzyme. It is suggested that these subunits are related to the assembly of RuBP carboxylase.     

Purification of ribulose-1, 5-bisphosphate carboxylase/oxygenase with high specific activity by fast protein liquid chromatography

A rapid procedure for the purification of ribulose-1, 5-bisphosphate carboxylase/oxygenase (rubisco) (EC 4.1.1.39) by fast protein liquid chromatography (FPLC) is described. Chloroplasts isolated mechanically from spinach leaves were used as the source of a stromal extract enriched in rubisco. By subsequent fractionation of this extract on ion-exchange FPLC, highly purified rubisco (sp act 2.10-2.76 mumol/mg protein X min) was obtained in less than 30 min. The high specific activity and excellent stability of the final preparation can be attributed to the use of chloroplasts as a starting material and the short time required for the chromatographic separation, both of which minimize proteolytic activity.     

Electrostatic fields at the active site of ribulose-1,5-bisphosphate carboxylase.

A macroscopic approach has been employed to calculate the electrostatic potential field of nonactivated ribulose-1,5-bisphosphate carboxylase and of some complexes of the enzyme with activator and substrate. The overall electrostatic field of the L2-type enzyme from the photosynthetic bacterium Rhodospirillum rubrum shows that the core of the dimer, consisting of the two C-terminal domains, has a predominantly positive potential. These domains provide the binding sites for the negatively charged phosphate groups of the substrate. The two N-terminal domains have mainly negative potential. At the active site situated between the C-terminal domain of one subunit and the N-terminal domain of the second subunit, a large potential gradient at the substrate binding site is found. This might be important for polarization of chemical bonds of the substrate and the movement of protons during catalysis. The immediate surroundings of the activator lysine, K191, provide a positive potential area which might cause the pK value for this residue to be lowered. This observation suggests that the electrostatic field at the active site is responsible for the specific carbamylation of the epsilon-amino group of this lysine side chain during activation. Activation causes a shift in the electrostatic potential at the position of K166 to more positive values, which is reflected in the unusually low pK of K166 in the activated enzyme species. The overall shape of the electrostatic potential field in the L2 building block of the L8S8-type Rubisco from spinach is, despite only 30% amino acid homology for the L-chains, strikingly similar to that of the L2-type Rubisco from Rhodospirillum rubrum. A significant difference between the two species is that the potential is in general more positive in the higher plant Rubisco. In particular, the second phosphate binding site has a considerably more positive potential, which might be responsible for the higher affinity for the substrate of L8S8-type enzymes. The higher potential at this site might be due to two remote histidine residues, which are conserved in the plant enzymes.     

Translational regulation of light-induced ribulose 1,5-bisphosphate carboxylase gene expression in amaranth.

The regulation of the genes encoding the large and small subunits of ribulose 1,5-bisphosphate carboxylase was examined in amaranth cotyledons in response to changes in illumination. When dark-grown cotyledons were transferred into light, synthesis of the large- and small-subunit polypeptides was initiated very rapidly, before any increase in the levels of their corresponding mRNAs. Similarly, when light-grown cotyledons were transferred to total darkness, synthesis of the large- and small-subunit proteins was rapidly depressed without changes in mRNA levels for either subunit. In vitro translation or in vivo pulse-chase experiments indicated that these apparent changes in protein synthesis were not due to alterations in the functionality of the mRNAs or to protein turnover, respectively. These results, in combination with our previous studies, suggest that the expression of ribulose 1,5-bisphosphate carboxylase genes can be adjusted rapidly at the translational level and over a longer period through changes in mRNA accumulation.     

Km values of ribulose-1,5-bisphosphate carboxylase of cassava cultivars

Ribulose-1,5-bisphosphate (RuBP) carboxylase activities of two cassava cultivars increased with leaf age but their K_m(CO₂) and K_m(RuBP) values remained relatively constant. K_m(CO₂) values of 16 cassava cultivars ranged from 7.8 to 14.0 μM CO₂, while K_m(RuBP) values varied from 7.5 to 24.8 μM RuBP. Differences in the K_m values could not be attributed to different physiological ages of plant material or to intravarietal variation, and are more likely to have been inherited. The results also showed that K_m values have potential applications in cassava systematics.     

Partition kinetics of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum

When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA).