RuBisCo active site

PHOTOAFFINITY LABELING OF THE RUBISCO ACTIVE-SITE AND SMALL SUBUNIT WITH 8-AZIDOADENOSINE 5 -TRIPHOSPHATE... 

Photoaffinity labeling of the Rubisco active-site Two lines of indirect evidence showed that binding of 8-N3ATP to the LSu of Rubisco takes place in the active-site. First, covalent modification of Rubisco by incorporation of the photoprobe reduced enzyme activity by 14.4%, an amount equivalent to the amount of [32p]8-N ATP incorporated into the LSu (data not shown). Second, phosphorylated effectors afforded a level of protection to the site which correlated with the apparent affinity of each for the active-site of Rubisco (data not shown). Photoaffinity labeling of the SSu was unaffected by active-site-directed effectors (data not shown). 

The X-ray structure of spinach Rubisco identifies 10 loop regions in the active-site (8). The major photolabeled peptide isolated by ion-exclusion and ion-exchange chromatography, and reverse phase HPLC corresponded to the Rubisco LSu tryptic peptide Val-42 to Arg-79 (Table 1), an active-site peptide (9). Two portions of the tryptic peptide Asp-95 to Lys-128, the peptide which comprises loop B in the crystal structure (8) were also recovered. Also recovered were two peptides (Table 1) which include residues in two other loop regions of the Rubisco active-site (8). The peptide Asp-202 to Arg-213 (loop 2 in ref. 8) is adjacent to the activator lysine (10). The peptide Gln-259 to His-267 is the N-terminal fragment of Gln-259 to Arg-285 the tryptic peptide which encompasses loop 4. Thus, all four of the photoaffinity labeled LSu peptides have been previously shown to be active-site peptides based on X-ray crystallography or chemical modification or by both techniques. 

Photoaffinity Labeling of the Rubisco Active-Site and Small Subunit with 8-... 

Figure 4.8 The active site in all a/p barrels is in a pocket formed by the loop regions that connect the carboxy ends of the p strands with the adjacent a helices, as shown schematically in (a), where only two such loops are shown, (b) A view from the top of the barrel of the active site of the enzyme RuBisCo (ribulose bisphosphate carboxylase), which is involved in CO2 fixation in plants. A substrate analog (red) binds across the barrel with the two phosphate groups, PI and P2, on opposite sides of the pocket. A number of charged side chains (blue) from different loops as welt as a Mg ion (yellow) form the substrate-binding site and provide catalytic groups. The structure of this 500 kD enzyme was determined to 2.4 A resolution in the laboratory of Carl Branden, in Uppsala, Sweden. (Adapted from an original drawing provided by Bo Furugren.)...

Rubisco exists in three forms an inactive form designated E a carbamylated, but inactive, form designated EC and an active form, ECM, which is carbamylated and has Mg at its active sites as well. Carbamylation of rubisco takes place by addition of COg to its Lys ° e-NHg groups (to give e—NH—COO derivatives). The COg molecules used to carbamylate Lys residues do not become substrates. The carbamylation reaction is promoted by slightly alkaline pH (pH 8). Carbamylation of rubisco completes the formation of a binding site for the Mg that participates in the catalytic reaction. Once Mg binds to EC, rubisco achieves its active CM form. Activated rubisco displays a Ai, for CO2 of 10 to 20... 

Substrate RuBP binds much more tightly to the inactive E form of rubisco (An = 20 nM) than to the active ECM form (A, for RuBP = 20 ixM). Thus, RuBP is also a potent inhibitor of rubisco activity. Release of RuBP from the active site of rubisco is mediated by rubisco activase. Rubisco activase is a regulatory protein it binds to A-form rubisco and, in an ATP-dependent reaction, promotes the release of RuBP. Rubisco then becomes activated by carbamylation and Mg binding. Rubisco activase itself is activated in an indirect manner by light. Thus, light is the ultimate activator of rubisco. 

FUNCTION RUBISCO CATALYSES TWO REACTIONS THE CARBOXYLATION OF D-RIBULOSE 1,5-BISPHOSPHATE, THE PRIMARY EVENT IN PHOTOSYNTHETIC CARBON DIOXIDE FIXATION, AS WELL AS THE OXIDATIVE FRAGMENTATION OF THE PENTOSE SUBSTRATE IN THE PHOTORESPIRATION PROCESS. BOTH REACTIONS OCCUR SIMULTANEOUSLY AND IN COMPETITION AT THE SAME ACTIVE SITE. 

The minimal functional unit (quite well conserved among all rubiscos) is a homodimer in which the active sites are located at the subunit interface. Residues from both subunits contribute to each active site, which is illustrated in Color Plate 8. All known forms (at present, four different types) consist of these basic dimeric units which are arranged into various larger multimer arrays—dimers, tetramers and even pentamers. The different forms of rubisco all have a common evolutionary origin and existing solid-state structures of the active sites are nearly superimposable. ... 

Before substrate binding can take place, rubisco must first be activated. This occurs via carbamylation (reaction with CO2) of an essential Lys residue . This promotes the binding of an essential Mg + ion after which the active site is complete. Rubisco can now recognize and bind the first substrate which is ribulose-P2 (D-ribulose 1,5-bisphosphate) . The substrate is bound to the Mg + ion via an inner-sphere coordination of the C2-carbony 1 and C3-hydroxyl groups which appropriately positions and activates the ribulose-P2 for subsequent reaction. Substrate binding causes a flexible loop to close over the active site which buries the active site deep within the protein and restricts access to a small channel just large enough for CO2 (and 02) . 

Central to the proposed mechanism for plant rubisco is a carbamoylated Lys side chain with a bound Mg2+ ion. The Mg2+ ion brings together and orients the reactants at the active site (Fig. 20-6) and polarizes the C02, opening it to nucleophilic attack by the five-carbon enediolate reaction intermediate formed on the enzyme (Fig. 20-7). The resulting six-carbon intermediate breaks down to yield two molecules of 3-phosphoglycerate. 

In most plants photosynthesis is also strongly inhibited by 02. This observation led to the discovery that 02 competes directly for C02 at the active site of rubisco in a process called photorespiration. Chloroplasts inhibited by oxygen produce glycolate in large amounts2823 as a result of the reaction of the intermediate enediolate ion formed in step b of Eq. 13-48 with 02... 

Figure 13-11 (A) Overview of the active site of spinach rubisco showing bound 2-carboxy-D-arabinitol 1,5-bisphosphate and Mg2+ and residues within hydrogen-bonding distance of these ligands. The hydroxyl groups at C2 and C3 of the inhibitor are in cis conformation. 269 Courtesy of Inger Andersson. (B) Structure of the inhibitor 2-carboxy-D-arabinitol 1,5-bisphosphate. A part of the carbamylated lysine 201 and the essential metal ion are also shown.

In this chapter, we present three approaches to address mechanistic issues with Rubisco mutants characterization of catalysis of partial reactions, analysis of side products, and subtle alteration of the active-site microenvironment by manipulation with exogenous reagents. 

The overall carboxylation or oxygenation of RuBP as catalyzed by Rubisco consists of discrete partial reactions illustrated in Fig. 1 (reviewed extensively in 1-3, 19). Because an active-site residue will not necessarily be involved in all catalytic steps, site-directed mutants devoid of overall activity may retain competence in one or more of the partial reactions. Independent of overall carboxylase activity, enolization of RuBP and turnover of the isolated six-carbon reaction intermediate can be assayed as distinct reactions, providing an avenue for discerning the particular step(s) preferentially facilitated by a given active-site residue. 

Another potential side reaction of the enediol(ate) intermediate is formation of the dicarbonyl compound, l-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, resulting from p-elimination of the Cl-phosphate due to improper stabilization and/or premature dissociation of enediol(ate) from the enzyme active site. This compound has been characterized by reduction with borohydride, oxidation with H2O2, complexation with o-phenylenediamine, and 13C-NMR (23, 34). The p-elimination product is not detected in reactions with wild-type R. rubrum Rubisco but is formed in substantial amounts with mutants in which the Cl-phosphate ligands are substituted, demonstrating the required role of these amino acid side chains in stabilizing the enediol(ate) intermediate (34-35). 

Figure 20.3. Structure of Rubisco. The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) comprises eight large subunits (one shown in red and the others in yellow) and eight small subunits (one shown in blue and the others in white). The active sites he in the large subunits.

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