Catalytic mechanism and regulation of Rubisco activity

Introduction

Fig.13: Variation of the βA-βB loop of the small subunit in various Rubisco enzymes. Large subunits are coloured blue, small subunits are yellow, and the βA-βB loops are red. A: Synechococcus sp. PCC 6301 (PDB 1RBL). B: Spinacia (PDB 8RUC). C: Chlamydomonas reinhardtii (PDB 1GK8). D:

Galdieria partita (PDB 1BWV, reproduced from Anderson and Backlund¹⁴³).

Introduction

Fig.14: Regulation of Rubisco activity by reversible carbamylation and inhibitor binding to carbamylated or non-carbamylated Rubisco. Rubisco activase and small molecule inhibitors control the activity of Rubisco. E and ECM signify non-activated and active Rubisco, respectively. ER and ECMI are inhibited forms (reproduced from Müller-Cajar et al.¹⁵¹).

Formation of the carbamate is followed by the rapid addition of Mg²⁺ to create the active ternary complex. This complex does not require the presence of small subunits¹⁵². In place of Mg²⁺, Rubisco can accommodate Mn²⁺, Fe²⁺, Ca²⁺, or Cu²⁺ as activating metal ions^153,154. The RbcL8RbcS8 Rubiscos contain eight catalytic sites. Both RuBP and various other effectors interact with the enzyme at a common site, the catalytic site. This results in the fact, that all of the compounds that influence the carbamylation state of the enzyme are competitive inhibitors of catalysis with respect to RuBP¹⁵⁵. Hatch and Jensen¹⁵⁶ classified effectors into two groups.

Positive effectors, such as NADPH and 6-phosphogluconate, enhance carbamylation.

Negative effectors, ribose-5-phosphate for example, favor the decarbamylated state. During Rubisco’s multistep catalytic reaction, protonation and oxygenation of the RuBP enediolate intermediate can result in the formation of isomeric pentulose bisphosphates, so-called misfire by-products¹⁵⁷. These include xylulose-1,5-bisphosphate (XuBP), 2,3-pentodiulose-1,5-bisphosphate (PDBP) and 3-ketoarabinitol-1,5-2,3-pentodiulose-1,5-bisphosphate (KABP)^157,158, whose formation results in an inactive, ‘closed’ enzyme that reactivates only slowly, limited by the spontaneous opening of the active site (Fig.14). The latter process is accelerated by the activity of another protein, Rubisco activase (Rca), a member of the AAA+ protein family, by an ATP-dependent mechanism (Fig.14)^159,160. Interestingly, the activity of Rubisco activase is controlled by redox-regulation in many plant species, which allows for indirect control of Rubisco activity in response to the redox status of the chloroplast stroma due to illumination. In many species in darkness and low light, carboxyarabinitol 1-phosphate (CA1P) is responsible for the low activity 30

Introduction

of Rubisco by binding to its activated form¹⁶¹. Presumably in the plant, as photosynthesis slows in shade or dark conditions, this intermediate analog stabilizes the activated state of the enzyme. CA1P is released by Rubisco activase¹⁶² after which it is rendered non-inhibitory by a specific, redox-modulated phosphatase^163,164. In the case of XuBP, a specific hydrolase, CbbY, cooperates with the action of Rca by degrading the released inhibitor, thereby preventing its rebinding¹⁶⁵. In the inactivated state, the enzyme can be also inhibited by its natural substrate RuBP, which can accumulate to high levels. One positive consequence of these naturally occurring stable binary states might be the protection of flexible elements from proteolysis during periods of low photosynthetic activity. Once Rubisco is activated, the stage is set for catalysis. The reaction mechanism is ordered, with RuBP binding before addition of the gaseous substrates CO2 or O2166. The catalytic process of carboxylation and oxygenation involve a sequence of analogous intermediates except for a final protonation that is lacking in the case of oxygenation (Fig.15).

Fig.15: Sequence of reactions catalyzed by Rubisco. RuBP: D-ribulose 1,5-bisphosphate, CKABP:

2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate, PKABP: 2-peroxo-3-keto-D-arabinitol-1,5-bisphosphate, 3PGA: 3-phospho-D-glycerate, 2PG: 2-phosphoglycolate (reproduced from Kannappan and Gready¹⁶⁷).

The initial enolization of RuBP is common to both pathways. CO2 or O2 then compete for the resulting enediol producing either a carboxyketone (CKABP, Fig.15) or a peroxyketone (PKABP, Fig.15), respectively. These ketones are hydrated, either in concert with the addition of the gases or subsequently. The hydrated ketones (gem-diol in Fig.15) then split 31

Introduction heterolytically between C-2 and C-3. In the case of oxygenation, this completes the reaction.

Carboxylation involves one further step: a proton must be added to the Si face of C-2 of the aci-acid (carbanion intermediate in Fig.15) produced from C-1 and C-2 of RuBP and the incoming CO2 molecule in order to produce the second molecule of 3PGA. The active site of Rubisco catalyzes all five reactions, i.e. enolization, gas molecule addition, hydration, C-C cleavage and protonation, without reopening at intermediate steps. The active site is formed from elements of the C-terminal barrel domain of one RbcL-subunit and the N-terminal domain of the second RbcL-subunit in the dimer. In the inactive enzyme, the site is open and accessible to activating cofactors and bisphosphate substrate¹²⁸. After formation of the essential carbamate and coordination of the Mg²⁺, RuBP binds and a series of loops close over the site to enfold and capture the bisphosphate^127,131. Closure of the loops brings together amino acids that are critical for catalysis and determine the fate of the substrate (Fig.16). The region of the barrel that comprises the active site is at the C-terminal end of the eight β-strands that form the core of this domain. The loops that connect the strand and helical elements of the barrel extend above and over the surface of the domain, contributing the amino acids that form the 1- and 5-phosphate binding sites and an extensive hydrogen bonding network with the sugar backbone of RuBP.

Fig.16: Cross-sectional view through the active site of tobacco Rubisco. Left: residues that contact the transition-state analoge CABP (silver) are shown in stick representation. Oxygen, nitrogen and phosphorus atoms are coloured red, blue and orange, respectively. The catalytic Mg2⁺ ion is shown as a green sphere. One of its ligands is the carbamylated lysine residue 201 (cyan). The active site is at the interface between two large Rubisco subunits (coloured brown and blue). Loop 6 (on the right) seals the binding pocket from the solvent. Right: open and closed conformations of Rubisco. The superposition of the crystal structures for the open (PDB 1EJ7) and closed (PDB 4RUB) states of Rubisco reveals conformational changes around the active site. In the closed conformation (brown and blue ribbons), the C-terminal peptide highlighted in cyan locks down loop 6 (from the right) on top of the active site indicated by CABP (stick representation), whereas in the open conformation (green and yellow ribbons) the C-terminal peptide is oriented away from the active site in a flexible conformation and loop 6 is detached (reproduced from Müller-Cajar et al.¹⁵¹).

32

Introduction

As mentioned above, in C3 plants, a number of serious limitations to the efficiency of photosynthesis are caused by the catalytic properties and regulation of Rubisco. There are two major factors affecting the carboxylase vs. oxygenase reaction catalyzed by Rubisco: the relative concentration of CO2 and O2 at the active site and the ability of the enzyme to discriminate between the two gaseous substrates. The latter one is expressed by the specificity factor SC/O = (k^Ccat/Kc)/(k^Ocat/KO), where k^Ccat, k^Ocat are the maximum velocities and KC, KO the Michaelis constants for CO2 and O2, respectively¹⁴⁵. Rubisco has very low substrate turnover rates of only ~3-10 CO2 molecules per second. The specificity factors vary among different Rubiscos, the lowest for Rhodospirillum rubrum (12). Moderate values are found in culture plants (e.g. Nicotiana tabacum with 82), but the highest are found in marine red algae (e.g.

Griffithsia monilis with 167). Calculations of a “perfect” Rubisco with an optimal catalysis rate and fixing exclusively CO2, suggest that this enzyme could maintain photosynthesis with 86%

less water loss, 35% less light, and 99% less protein investment in Rubisco¹⁶⁸. Therefore, one strategy to increase photosynthetic carbon fixation would be to generate C3 crop plants expressing Rubisco with high specificity factor and catalytic rate^169-171. Attempts to introduce rbcL and rbcS operons for the high specificity factor red-type Rubiscos of Galdieria sulphuraria and Phaeodactylum tricornutum into the plastid genome of tobacco have only partially succeeded¹⁷². Whilst the transgenes directed the synthesis of transcripts in abundance, the subunits of these foreign Rubiscos were insoluble, indicating that one or more processes associated with the folding and/or assembly of the red-type Rubisco will need to be understood much better and perhaps translated into chloroplasts if this type of Rubisco transplantation is to be successful.