RUBISCO: structure and mechanism.

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Annu. Rev. Biophys. Biomol. Struct. 1992.21:119-143. Downloaded from www.annualreviews.org by Rice University on 07/19/13. For personal use only.

RUBISCO: Structure and Mechanism Gunter Schneider, Ylva Lindqvist, and Carl-Ivar Branden

Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Box 590, S-751 24 Uppsala, Sweden KEY WORDS:

ribulose-l,5-bisphosphate carboxylase, X-ray crystallography, photosynthesis, photorespiration, protein engineering

CONTENTS PERSPECTIVES AND OVERVIEW........................................................................................

1 19

PHYSIOLOGICAL AND GENETIC ASPECTS..........................................................................

120 120

Photosynthesis and Photorespiration......................................................................... Synthesis and Assembly . . . . . ................. . . ... ...... . .... . .. . . . .. . . ......... . . . . . . . . . . .......... . . .. CHEMICAL MECHANISM .................................................................................................

Activation-the Ternary Complex of Enzyme, CO" and Mg(II) ............................. Overall Carboxylation Reaction . .... . . .... .. ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . ...... . ..... . . . . . . . . . . . . . . . Partial Reactions and Reaction Intermediates...........................................................

.

..

121

122 122

123

124

THREE·DIMENSIONAL STRUCTURE..................................................................................

126

Overall Structure of Rubisco as Revealed by Protein Crystallography ...................... Structural Differences Small Subunit................................................................................................ The Active Site . ............... . . . . . . . . ....... . . . . . . . . . . . ...... ...... . . . . ... ........... . . . . . . . . . . ............... . . . . . . . . Binding of Phosphorylated Compounds to Rubisco.................................................... Conformational Changes During Catalysis ...............................................................

126

STRUCTURE AND FUNCTION...........................................................................................

1 36 1 36 137

Activation............................................... .. . . .................. ................ ..... . . . . .................. Enolization .......... . . . . . . . . . . ..... . . . . . . . . . . . . . . .......... . . . . . . . . . . . . ..... . . . . . . . . . . . ........ . . . . . . . . .................. Carboxylation .......................................................................................................... SUMMARY AND PROSPECTS ........................................................................... .................

129

130 1 32 135

138

140

PERSPECTIVES AND OVERVIEW

Solar energy that is captured and converted to chemical energy during photosynthesis sustains almost all lifeforms on earth. Most of this energy is stored by converting CO2 to polymerized sugar molecules from which most nonphotosynthetic organisms ultimately obtain the energy needed to drive their cellular reactions. The initial step in this photosynthetic fixation of CO2, the carboxylation of ribulose- l ,5-bisphosphate (RuBP), is 119 1056-8700/92/06 10-0119$02.00

120

SCHNEIDER, LINDQVIST & BRANDEN

catalyzed by the enzyme ribulose- l ,5-bisphosphate carboxylase oxygenase (Rubisco). This enzyme is responsible for the annual net fixation of

1011

tons of CO2 from the atmosphere to the biosphere. This amount can be compared to our annual net consumption of crude oil, which is about 3

x

109 tons.

Rubisco is perhaps the most abundant protein on earth; it is certainly the most abundant enzyme: up to

50%

of leaf proteins in plants are


Rubisco. This large amount does not result primarily from the enormous task the enzyme has to carry out, but rather reflects the catalytic inefficiency of Rubisco as a catalyst, which is manifested by a turnover number of 3

S-I.

Not only is the enzyme slow, it also catalyzes a competing oxygenase reaction that leads to loss of energy by photorespiration. In this reaction, O2 instead of CO2 is added to RuBP, yielding phosphoglycolate as one of the oxidation products. Phosphoglycolate is metabolized in the glycolate pathway, which eventually produces CO2 and energy in the form of heat. The net efficiency of photosynthesis is reduced by up to

50%

by this

photorespiratory pathway, which also severely affects a plant's water-use efficiency and nitrogen budget. For these reasons, genetic redesign of Rubisco with the aim of con structing transgenic plants with improved photosynthetic efficiency and thereby increased agricultural productivity has attracted a lot of interest. Detailed kinetic studies of the catalytic reactions of Rubisco have been carried out during the past two decades and are summarized in several excellent reviews

(10, 47, 48, 79, 96). This review describes recent advances

in our knowledge of the molecular details of the enzyme's function that have emerged from genetic and X-ray structural studies.

PHYSIOLOGICAL AND GENETIC ASPECTS Photosynthesis and Photorespiration Rubisco is one of the key enzymes in the carbon metabolism of plants. The enzyme catalyzes the initial step in CO2 fixation, the carboxylation of RuBP, yielding two molecules of phosphoglycerate. RuBP, the initial CO2 acceptor, is regenerated in the Calvin cycle, and the fixed carbon is incorporated into carbohydrates such as sucrose and starch. The oxy genation of RuBP results in the formation of one molecule of phos phoglycerate, which can be metabolized in the Calvin cycle, and one molecule of phosphoglycolate. To salvage some of the carbon of phos phoglycolate, C-3 plants have developed the glycolate pathway, in which some of the phosphoglycolate is converted to phosphoglycerate. This


RUBISCO

121

rescue pathway consumes considerable amounts of energy, however, and 50% of the carbon is still lost as CO2, Carboxylation and oxygenation of RuBP occur at the same catalytic site of Rubisco; both gaseous substrates compete for the second substrate, RuBP. Therefore, the ratio of carboxylation towards oxygenation is influ enced by the relative concentrations of CO2 and O2, Higher CO2 con centrations result in more efficient photosynthesis with faster production of biomass (52). The present efforts to increase the concentration of atmo spheric CO2 might result in higher agricultural yields on a global scale. A doubling of atmospheric CO2 concentration is expected to increase agricultural yields by approximately 33% (72). Rubisco activity is highly regulated in vivo. A wealth of experimental data has accumulated showing that regulation operates at transcriptional as well as translational and posttranslational levels. Andrews & Lorimer (10) and Gutteridge & Gatenby (48) recently summarized the complicated patterns of Rubisco regulation. Synthesis and Assembly

Rubisco from higher plants and most photosynthetic microorganisms is built up of two types of subunits, large (L chain, M 52,000-55,000) and small (S chain, M 13,000). The holoenzyme is a complex of eight L and eight S subunits (LsSs Rubisco) forming a molecule with 422 symmetry (13), The only well-established exceptions from this quaternary structure are the enzymes found in the photosynthetic bacteria Rhodospirillum rubrum (121, 138) and Rhodobacter sphaeroides (44). These organisms contain a simpler type of Rubisco that consists of only two large subunits (L2 Rubisco). Interestingly, R. sphaeroides also expresses an LsSs Rubisco (44). The genes for the LgSg Rubisco molecule from higher plants are present in two different genomes. The gene for the L subunit is part of the chlo roplast genome, whereas the S subunits are coded by the nuclear genome. Each chloroplast DNA molecule has only one gene for the L subunit. Even though each chloroplast contains several DNA molecules and each cell contains hundreds of chloroplasts, a nonhybrid plant will have only one unique sequence for all L subunit proteins. In contrast, a family of genes on the nuclear genome codes for the S subunits. These genes are strongly homologous. For example in A rabidopsis thaliana, four genes have been identified that show more than 90% sequence identity in their gene pro ducts (75). The S-subunit genes are differentially expressed in different cell types, but specific functional roles for individual genes have not been demonstrated (31, 102). Amino acid sequences are known for about 20 =

=


122


different L chains from various species and 35 different S chains, mostly deduced from the corresponding genes or cDNA sequences. S subunits are translated as preproteins that contain an extra peptide of about 50 residues at the amino end of the polypeptide chain. This peptide directs the S subunit into the chloroplast, where it is cleaved off after passage of the preprotein through the membranes (29). Assembly of the L subunits with mature S subunits inside the chloroplast is mediated by a chaperone protein, Rubisco binding protein (16, 10 1, 1 17). This protein, which is present in large amounts in plants, belongs to the chaperonin family, one member of which is the classic chaperone protein GroEL from Escherichia coli (43). The Rubisco binding protein and GroEL show about 50% amino acid sequence identity (56). The chaperonin protein is believed to prevent improper aggregation of L sub units by protecting exposed hydrophobic surfaces during the last stages of the folding or assembly process (34, 45). The gene for the R. rubrum L subunit has been cloned and expressed in E. coli (104, 130). Functional L2 molecules are obtained in high yield (Il l ), and this system has been extensively used for site-directed mutagenesis experiments to probe the function of critical residues in the active site (50, 51, 53, 55, 77, 78, 100, 107, 131, 132, 142). In contrast, expression in E. coli of Rubisco L- and S-subunit genes from higher plants yield insoluble aggregates with no catalytic activity (40, 42). The subunits do not assemble into functional L sS s molecules, presumably because of lack of proper chaperone proteins. However, L and S subunits from cyanobacteria obtained by coexpression in E. coli of the corresponding genes assemble into catalytically competent L sSs molecules. In these cyanobacteria, the genes for Rubisco L and S subunits are on the same operon, and apparently assembly does not require cyanobacterial chaperone proteins. This is the system of choice for making and analyzing mutations in the S subunit (28, 38, 4 1, 46, 70, 139). CHEMICAL MECHANISM

Activation-the Ternary Complex of Enzyme, CO2, and

Mg(IJ)

A common feature of all Rubisco molecules analyzed so far is a chemical modification step necessary to convert the enzyme from its inactive to its active form. The activation process consists of the formation of a car bamate group by reaction of a CO2 molecule with the s-amino group of a lysine residue at the active site (80, 87). This activator CO2 molecule is separate from the CO2 molecule that becomes incorporated into RuBP during catalysis (81,95). Formation of the carbamate is followed by rapid


RUBIsea

1 23

binding of Mg(II), resulting in the active ternary complex-enzyme, CO2, and Mg(II) (81, 84). In the ternary complex, the Mg(II) ion can be replaced by several different metal ions such as Mn(II), Fe(II), Ca(II), Cu(lI), Co(II), and Ni(II). However, the degree of catalytic activity depends both on the type of metal and the type of Rubisco. In L2 Rubisco, catalytic activity has been observed upon replacement of Mg(Il) by Fe(Il), Ca(H), and Cu(II) (26, 27, 1 06, 1 l 3, 116, 133). In the L8S8 enzyme, activity has been observed with Mg(Il), Ni(II), Co(Il), Fe(II), Mn(II), and Cu(lI) ( 19, 20, 26, 27, 146, 147). A very interesting observation is that the metal bound at the activator site can influence partitioning between carboxylation and oxygenation. Rubisco molecules from R. rubrum activated with Co(Il) (26, 1 16) and from spinach activated with Cu(II) (19) are active as oxygenases, but lack carboxylase activity. This indicates an intimate involvement of the metal ion in at least one catalytic event: the addition of the gaseous substrate, CO2 or O2, respectively, to RuBP. Formation of the carbamylated ternary complex is accompanied by changes in the chemical properties of the enzyme. Carbamylation affects the reactivity and nucleophilicity of certain lysine residues at the active site, reflected by different chemical labeling patterns observed in experi ments with affinity labels (39, 57) or group-specific reagents (54). Also, the affinities for the substrate RuBP (66) and other phosphorylated com pounds (68, 94) are influenced by the carbamylation state of the enzyme. Overall Carboxylation Reaction

This review focuses on the carboxylation reaction of Rubisco, which is far better understood than the oxygenation reaction. Carboxylation of RuBP is a complicated reaction that involves a series of events and several intermediates. A preliminary mechanism for the carboxylation reaction, which has subsequently been modified, 'was first proposed by Calvin (21). The overall reaction can be divided into a number of individual steps (Figure 1). This overall scheme is supported by many isotope-labeling experiments. Lorimer (79) provides an excellent summary of these data. The first step in this mechanism is the enolization of RuBP, resulting in the 2,3 enediol(ate). Carboxylation at the nucleophilic center at C-2 creates a six-carbon intermediate 2-carboxy-3-keto-arabinitol 1,5 bisphosphate (3-keto-CABP), which undergoes hydration to the gem diol form. Depro tonation of the gem diol at 0-3 of the six-carbon intermediate initiates carbon-carbon cleavage that results in one molecule of 3-P-glycerate and the C-2 carbanion form of another 3-P-glycerate molecule. The carbanion is then stereospecifically protonated at C-2, yielding the second 3-P-glycer ate molecule. The release of products completes the catalytic cycle.


124


IEnolisation and Carboxylation I IHydration II C-C cleavage and protonationl

{

° II

C U

fHzO®

C=O I

\

H-C-OH � I

H-C-OI{ I rn..

CH20W

CH,o®

fH,o® '- C-OH

I

-? fHaO®

O=C-C-OH

HO-C-�-

• C-OH I

�

I H20 c==o � I

H-C-OH

H-C-CH

I

I

CH20®

CHaO®

0, *0 C

I H-C-OH

�zO® Figure 1

Mechanism of the carboxylation reaction.

Partial Reactions and Reaction Intermediates 2,3 ENEDIOL(ATE) Enolization of bound RuBP is considered to be the very first step in the catalytic cycle. Tn fact, this step is common to both the carboxylation and oxygenation reactions. Enolization, which is initiated by abstraction of the C-3 proton of the substrate, leads to formation of the 2,3 enediol(ate) of RuBP as the first intermediate during turnover. This partial reaction, the exchange of the proton at C-3 of RuBP, can be followed by the wash in of solvent 3H into C-3 of RuBP and the wash out of[3-3H]RuBP into the solvent (119, 136).

ENOLIZATION OF RuBP: THE

CARROXYLATION: 3-keto-CABP At the stage of the 2,3 enediol(ate), the reac tion proceeds either towards carboxylation or oxygenation. In both reac tions, the gaseous substrates, CO2 or O2, respectively, react with the C-2 carbon atom of RuBP. Electrophilic attack of CO2 on the C-2 carbon atom yields the six-carbon intermediate, 3-keto-CABP. After acid quenching of the carboxylation reaction, the intermediate can be trapped by borohy dride reduction to the corresponding 2' carboxypentitol bisphosphate (120). The borohydride reduction of the intermediate occurs only in free solution, not when bound to the enzyme. This probably results from the fact that the six-carbon intermediate exists on the enzyme predominantly as the hydrated C-3 gem diol, which cannot be reduced by borohydride (83). One of the unique features of the carboxylation reaction is that the

RUBTSCO

125


intermediate 3-keto-CABP can be isolated and is surprisingly stable (83, 110). By acid quenching of the reaction, sacrificing large amounts of enzyme, the intermediate can be isolated and stored at - 80°C for months. At room temperature, 3-keto-CABP is reasonably stable: it decarboxylates with a half-time of approximately I h (83). HYDRATION AND CARBON·CARBON BOND CLEAVAGE: THE GEM OWL On the enzyme, the intermediate 3-keto-CABP exists predominantly in the gem diol form, resulting from the addition of a water or hydroxide molecule to the C-3 carbon atom. The 0-3 oxygen of RuBP is completely retained during carboxylation (82, 135). Thus, the hydration step is either kinetically reversible and/or stereospecific. Abstraction of a proton from the gem diol initiates carbon-carbon bond cleavage between C-2 and C-3, yielding one molecule of 3-phospho-glycerate and the C-2 carbanion of 3-phospho glycerate. The isolated 3-keto-CABP can be added back to the enzyme and is then hydrolyzed to products. However, Rubisco catalyzes the hydrolysis of 3keto-CABP at rates that are only 3% of the maximal rate of carboxylation. This discrepancy between rates of carboxylation and hydrolysis led Cleland (30) to question the kinetic competence of 3-keto-CABP as a true reaction intermediate. He suggests a modified mechanism in which the car boxylation and the hydration steps occur in a concerted reaction, resulting in the gem diol. Therefore, in his proposal, 3-keto-CABP is not a true reaction intermediate. However, other effects might cause the observed discrepancy in reaction rates (see below), and in our opinion, the exper imental cvidence for 3-kcto-CABP as a reaction intermediate (summarized in 10) is compelling. The availability of the six-carbon intermediate provides another partial reaction for probing the functional defects of site-directed mutants. Mutant Rubiscos deficient in the overall carboxylation reaction might be able to catalyze the hydrolysis of the six-carbon intermediate to products. One can thus distinguish between mutants deficient in the enolization reaction, verificd by the wash in or wash out experimcnts, or mutants deficient in one of the subsequent steps of catalysis. PROTONATION: THE C·2 CARBANION The carbon-carbon bond cleavage of the gem diol results in the formation of one molecule of 3-phosphoglycerate and one molecule of the C-2 carbanion of 3-phosphoglycerate. However, no firm experimental evidence for the existence of the C-2 carbanion as an intermediate has been obtained so far. Stereochemical considerations ( 10) suggest that the enzymic base donating the proton to the C-2 carbanion is different from the groups involved in proton-proton transfer during the earlier enolization-carboxylation steps of catalysis.

126


THREE-DIMENSIONAL STRUCTURE

The Overall Structure of Rubisco as Revealed by Protein


Crystallography

Because of the central role of Rubisco in the carbon cycle, several groups are engaged in crystallographic studies of this enzyme from various sources. Crystallographic studies of Rubisco were first reported for the enzyme from tobacco (12, 13). These low resolution studies indicated that the molecule was a two-layered structure with 422 symmetry. The studies also showed a central channel running through the molecule. However, the crystals used were not sufficiently well ordered to allow structure determination to high resolution. Subsequently, preliminary crys tallographic data were reported for the homodimeric enzyme from R. rubrum (25, 63, 122, 123) and for the LgSg molecule from tobacco (14, 137), spinach (1, 3, 15), potato (65), A lcaligenes eutrophus ( 18, 109), Chromatium vinosum (103), and Synecococcus (105). Following the initial reports, the structure of the nonactivated R. rubrum enzyme was solved to 2.9-A resolution ( 125). This study revealed the two domain structure of the L subunit, with one smaller N-terminal domain and a larger C-terminal domain folded as an eight-stranded parallellY./f3 barrel, and also located the active site in the subunit interface at the C terminal end of the f3 strands in the IY./f3 barrel. The model of the non activated enzyme from R. rubrum has now been refined to I .7-A resolution (126). The structures of several different complexes of Rubisco from R. rubrum have also been reported (89-92). Chapman et al (22, 23) reported preliminary descriptions of the non activated LgSg enzyme from tobacco at 3.0-A and 2.6-A resolution. Anders son et al (2) described the active site of Rubisco based on the structure of nonactivated R. rubrum enzyme and activated spinach Rubisco complexed with 2-carboxy arabinitol 1,5 bisphosphate (CARP), an analog of the six carbon intermediate. Knight et al (73) described the fold of the small subunit that in the work by Chapman et al (23) had not been correctly determined. Knight et al (74) provided a complete description of the spinach Rubisco structure refined at 2.4-A resolution. THE L SUBUNIT The catalytic activity of Rubisco resides on the L subunit, the structure of which is very similar in L2 and LgSg Rubisco (124) despite the low amino acid sequence identity, 28 %. The L subunit consists of two clearly separated domains (Figure 2). The N-terminal domain, residues 1150, J is folded into a central mixed five-stranded f3-sheet with two a-helices I

All sequence numbering is based on the amino acid sequence of Rubisco from spinach. In

the case of the L subunits, this is the unified numbering system as suggested in Schneider et al (124).


RUBISCO

Figure 2

127

Schematic view of the L, Rubisco molecule from Rhodospirillum rubrum. One of

the two L subunits is shown in gray. Bound substrate, ribulose bisphosphate, is included.

on one side of the sheet. The larger C-terminal ex!f3 barrel domain consists of amino acids 151--475. This structural motif has been observed in many functionally nonrelated enzymes with completely different amino acid sequences (36). Eight consecutive f3ex units are folded such that the f3-strands form the core of a barrel surrounded on the outside by the eight IX-helices. The active site is located at the carboxyl side of the f3-strands; several of the last residues in the f3-strands, or the first residues in the loops between the strands and helices, are involved in catalysis. The L2 dimer has the shape of a distorted ellipsoid with dimensions 45 x 70 x 105 A and tight inter actions between the subunits. The core of the molecule is made up of the C-terminal domains from both subunits. The contacts at the interface between these domains involve interactions of the loops between the car boxyl ends of the f3 strands and the (X-helices from the (X!f3 barrels across the local two-fold axis of the molecule. The second contact area between the subunits involves the active site at the carboxy terminii of the f3 strands in the IX/f3 barrel of the C-terminal domain. This side of the barrel is partly covered by the N-terminal domain of the other subunit, and residues from both domains contribute to the formation of the active site. This interaction area includes an intricate THE L, DIMER: THE MINIMAL FUNCTIONAL UNIT

128



network of charges and hydrogen bonds involving conserved sidechains. Replacing one of these, Lys175, with an aspartate sidechain (78) disrupts the balance of charges, and the repulsion prevents dimerization. In addition, site-directed double mutant experiments have shown that the active site is located at the interface between subunits (77). Thus, because of this subunit packing, the dimer of large subunits is the minimal func tional unit. THE S SUBUNIT The 123 amino acids of the small subunit are arranged in a four-stranded anti-parallel tJ-sheet covered on one side by two a-helices. The amino-terminal residues form an arm of irregular structure that extends to a neighboring small subunit and forms the interaction area between small subunits that form S4 clusters. Structure determination of the spinach enzyme revealed deviations from the published sequence (93).

The LgSg molecule is shaped like a cube with rounded edges and sides of approximately l lO A each. The fundamental unit of the Lg part of the molecule is the L2 dimer. Each small subunit binds in a deep crevice formed between the tips of two adjacent elongated L2 dimers at each end of the LgSg molecule. Four faces of the cube-shaped molecule are thus formed from pairs of adjacent L2 dimers, whereas the remaining two faces are formed by the S4 clusters. The interaction areas between the small and the large subunits are quite extensive in LgSg Rubisco and involve several regions of residues that are conserved in all small subunits. Some of these have been probed in the Anabena eutrophus enzyme with mutations (38) that give subunits that do not assemble with the L subunits to form functional LBSB molecules. One of these mutations (Glu13-Val) disrupts a network of ion-pair interactions and would bury an unbalanced positive charge, thus preventing assembly. Another mutant (pro73-His) would result in burying a polar side chain in an otherwise very hydrophobic environment. Thus, it is not surprising that this mutant does not forin a stable holoenzyme. The surface of the L2 dimer in the spinach LsSs enzyme is somewhat less hydrophobic than expected (74) for a molecular entity involved in subunit interactions. Its surface is more like that of a monomer or a fully assembled oligomer, perhaps indicating an intermediate role for the dimer in the assembly process. This observation is also true for the (L2)4 core of large subunits. Nevertheless, researchers have been unable to isolate L subunits from plant Rubisco without concomittant aggregation and/or pre cipitation (67, 69, 118, 145), probably because of the exposure of highly hydrophobic patches on the surface of the L subunits upon release of S subunits. In contrast, in Rubisco from cyanobacteria in which the subunits can be easily separated to give isolated S subunits and stable (L2)4 cores THE L,S, MOLECULE

RUBISCO

129


(5, 6, 1 1, 6 1,62, 67, 140), several of these conserved hydrophobic residues are changed to polar or even charged side chains. The (L2)4 core Rubisco binds small subunits and reassembles to form a species indistinguishable from the native, undissociated enzyme. This phenomenon has been explored by constructing Rubisco hybrids with (LZ)4 from one species and S subunits from another species (7, 9, 61, 144). The general conclusion from these experiments is that the hybrids show the character of the parent (L2)4 rather than the parent small subunit. In particular, they exhibit the

same carboxylase/oxygenase ratio as the parent (LZ)4. Structural Differences a Function of the Small Subunit

The small subunits profoundly influence the catalytic properties of LgSg Rubisco (5, 6, 61, 62, 67, 140). The Lg core of the enzyme retains a small intrinsic activity in the absence of small subunits (4). Structural studies (2, 22, 74) have shown that the small subunits do not contribute any residues to the active site. Thus, the dramatic effect of the small subunits on catalytic activity must be exerted by inducing long-range effects in the active site through interactions remote from the active site. Schneider et al ( 124) discussed how this might be accomplished, showing that the position of helix 8 with respect to the rest of the a/P barrel is different in the L2 versus the LgSg type of Rubisco. These structural differences extend to loop 8, which forms one of the phosphate binding sites in the active site, and to helix 7 and loop 7, which are also part of the active site and contain a strictly conserved serine residue involved in inhibitor, substrate, and prod uct binding (2, 89, 90, 92). These structural differences could be the basis for the higher catalytic efficiencies of the LgSg enzymes compared to R. rubrum Rubisco. The difference in structure is caused by the binding of small subunits that form extensive interactions with a-helix 8 and parts of loop 8. The position adopted by a-helix 8 in the R. rubrum enzyme would be sterically hindered in the LgSg enzyme because of overlap with the small subunit. This conformational difference is also reflected in the C-terminal region of the L chain, which forms a helical extension to the a/P barrel. An intrinsic difference between the R. rubrum and the plant enzyme in this part of the structure is that the L2 enzyme has two additional a-helices. The C-terminal residues (459-466) of Rubisco from R. ruhrum show a remote sequence homology to residues lJ-20 of the small subunit of LgSg Rubisco. A possible function of these residues as a substitute for the small subunit in L2 Rubisco was tested by truncation of the polypeptide chain at position 458 ( lIS) or 460 (98). The truncated chains form a mutant protein with almost undisturbed activities, showing that these residues are not essential for proper function. Truncation at positions 431 or 423 (115),


130


which removes helices aG, aH, aI, and aI, leads to insoluble protein aggregates because of the exposure of a hydrophobic patch at the surface of the mutant protein. Curiously, truncation at position 441 leads to the formation of an Ls Rubisco, different in packing from the (L2)4 core of the higher plant enzymes. Disruption of the hydrophobic patch by site-directed mutagenesis prevents the formation of the Ls particles (115). Another species difference involves the N-terminal residues. The first a helix in the R. rubrum enzyme is absent in the spinach enzyme. In the bacterial enzyme, this a-helix covers one side of the N-terminal fJ-sheet, which is thus more exposed in the spinach enzyme where the corresponding residues have the conformation of an extended chain. Differences in the N-terminal region might be of interest with regard to catalysis. Removal of the eight first residues in spinach (60) and wheat (49) Rubisco does not affect catalytic activity, whereas removal of six additional residues drastically reduces catalytic activity. The crystal structure of the spinach enzyme (74) shows that the side chain Phe13 is buried between the small subunit and the large subunit, causing extensive interactions. The dramatic effect on activity following removal of residues 8-14 in the large subunit indicates that the interactions in this area are important to maintain a catalytically competent enzyme. The Active Site

The active site of Rubisco is located at the interface between the C-terminal domain of one subunit and the N-terminal domain of the second subunit (2, 22, 125). As in all a/fJ barrel enzymes known so far, the active site is found at the carboxy ends of the fJ-strands of the barrel. Residues in the loops from the p-strands to the a-helices of the barrel contain most of the conserved amino acid side chains essential for substrate binding and catalysis. Loop 1 contains the sequence LysI75-ProI76-Lysl77, which is con served in all Rubisco molecules. In all Rubisco structures determined at sufficiently high resolution, the proline peptide bond is in the cis-con formation. Lys175 has been identified as an active-site residue through affinity labeling (58), crystallography (2, 23, 89, 125), and site-directed mutagenesis (55). The side chain of Lysl77 also points into the active site and interacts with the side chains of Glu60 and Glu204. Both lysine residues are part of a complicated network of charges and hydrogen bonds, involving residues Glu60, Lys175, Lysl77, Asp203, and Glu204. The disruption of this deli cate balance of charges by site-directcd mutagenesis severely affects tertiary and quaternary structure. Substitution of Asp203 with Asn induces local and global conformational changes (E. S6derlind, G. Schneider & S.


RUBISCO

131

Gutteridge, unpublished results), whereas the replacement of Lysl 75 with an aspartic acid side chain prevents the formation of dimers (78). Loop 2 also contains several conserved amino acid residues, Lys201, Asp203, and Glu204. These three amino acids form the activator site. The side chain of Lys201 becomes carbamylated during activation, and the carbamate, together with the side chains of Asp203 and Glu204, forms the metal-binding site (2, 91, 92). Loops 3 and 4 arc involved in subunit-subunit interactions, but do not contribute any residues directly involved in catalysis or substrate binding. In the enzyme from R. rubrum, Asp245 forms a bifurcate hydrogen bond to the main-chain nitrogen atoms of residues 113 and 1 14 at the N-terminal end of helix etC in the N-terminal domain of the second subunit (126). Loop 5 contains two conserved histidine residues, His294 and His298. His294 is close to the bound substrate and inhibitor and has been impli cated in catalysis (2, 74, 92). The function of His298 has been probed using site-directed mutagenesis. Substitution of this side chain by Al a produces a mutant enzyme with 40% activity (107), demonstrating that this residue is not essential for catalytic activity. A conserved arginine residue, Arg295, within this loop is involved in the binding of one of the phosphate groups of CABP (2, 90) or RuBP (92). Loop 6 is a flexible loop that is disordered in the crystals of nonactivated Rubisco from R. rubrum (125, 126). In those structures, where this loop is ordered, it has been observed in both an open an

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The Rubisco subunit binding protein.

Subunit interactions of Rubisco activase: polyethylene glycol promotes self-association, stimulates ATPase and activation activities, and enhances interactions with Rubisco.

Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides.

Electron microscopy of the complexes of ribulose-1,5-bisphosphate carboxylase (Rubisco) and Rubisco subunit-binding protein from pea leaves.

Influence of salicylic acid on rubisco and rubisco activase in tobacco plant grown under sodium chloride in vitro.

Rubisco decrease is involved in chloroplast protrusion and Rubisco-containing body formation in soybean (Glycine max.) under salt stress.