Harry Roy

Rubisco assembly: a model system for studying the mechanism of chaperonin action.

The chloroplast enzyme Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) catalyzes the carboxylation of ribulose bisphosphate in the Calvin cycle and the oxygenation of the same substrate in the photorespiratory pathway. Because these two competing reactions determine the net photosynthetic yield of many higher plants, it is important to understand their mechanisms. This requires not only a model of Rubisco atomic structure, but also experimental systems that allow the structure to be manipulated by in vitro mutagenesis. The requirement for a structural model has been fulfilled recently (Chapman et al., 1987, 1988; Andersson et al., 1989). Unfortunately, however, in vitro mutagenesis of higher plant Rubisco has not been possible because the subunits of higher plant Rubisco, either when dissociated from each other chemically in vitro (Voordouw, Van der Vies, and Boumeister, 1984) or when synthesized in Escherichia coli (Bradley, Van der Vies, and Gatenby, 1986), fail to assemble into active enzyme. Severa1 groups have attempted to overcome this problem by studying Rubisco assembly from either native subunits (Roy, Cannon, and Gilson, 1988a), prokaryotic subunits expressed in E. coli (Gatenby, 1988), or alga1 subunits (Newman and Cattolico, 1988). These studies indicate that the post-translational assembly of Rubisco holoenzyme in both prokaryotes and eukaryotes is a complex process that may require helper proteins called “chaperonins” (Gatenby, 1988; Hemmingsen et al., 1988; Roy and Cannon, 1988; Roy et al., 1988a; Ellis, Van der Vies, and Hemmingsen, 1989). Chaperonins are functionally analogous to other molecular chaperones such as nucleoplasmin, which is involved in nucleosome assembly, and the bovine microsomal binding protein, which participates in immunoglobulin transport (Pelham, 1986; Ellis et al., 1989). The molecular chaperones, including the chaperonins, are supposed to interact transiently with other proteins, promoting assembly of those proteins into functional complexes. The molecular chaperones do not form part of the final structure of the protein complexes whose assembly they promote. In this article I review recent experiments that persuasively support the idea that chaperonins are required for Rubisco assembly.

Rubisco: Assembly and Mechanism

Ribulose-bisphosphate carboxylase/oxygenase (Rubisco, E.C. 4.1.1.39) is unique to photosynthetic metabolism. Two intensively studied aspects of Rubisco physiology are covered in this chapter, its post-translational assembly and its mechanism of action. Bacterial Rubisco can be assembled in vitro and in bacterial hosts but, as yet, assembly in vitro of higher-plant Rubiscos has not been reported. This focuses attention on the assembly pathway for higher plant Rubisco, which has been known for some time to be related to the presence of molecular chaperones in chloroplasts. Analysis of mutants, transformation of plants and bacteria with chloroplast chaperones, and the development of in vitro translation and assembly systems based on chloroplast extracts, have been directed at resolving this problem. It appears from these data that certain bacterial chaperones do not interfere with the assembly of higher plant Rubisco. As in cyanobacterial systems, the absence of S subunits leads to the accumulation of L8-like particles whose subunits can later be recruited to form Rubisco. Subtle differences between the way S subunits assemble with higher-plant and cyanobacterial L8-like particles suggest that this process may be concerted with assembly of L8 in the case of the higher-plant enzyme. The catalytic mechanism of Rubisco depends on two co-factors; a divalent metal ion, usually Mg2+ and a CO2 molecule that carbamylates a specific lysyl residue, K201, in the active site. This carbamate plays a crucial role in initiating catalysis by abstracting the C3 proton of ribulose bisphosphate and it may also act as a general-base catalyst for succeeding steps. Sofar, Rubisco’s use of a carbamate as a base appears to be unique among enzymes. The catalytic sequences of both the carboxylation reaction, and the oxygenation reaction that competes with it, proceed through multiple steps, each of a complexity rivaling that of the complete reaction of many other enzymes. The structure of the active site must change subtly between steps. Selectivity between CO2 and O2, of paramount importance to photosynthetic efficiency, is determined by the relative reactivity of the enediol(ate) form of the substrate for the two gases.

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

Abstract Pea leaves, supplied with [ 35 S]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.

Ribulose bisphosphate carboxylase assembly: what is the role of the large subunit binding protein?

Abstract Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), is a key enzyme in both photosynthetic carbon fixation and photorespiration. Genetic manipulation of this enzyme to increase its carboxylase activity could lead to substantial increases in net photosynthesis in several important crop plants. Unfortunately, the higher plant enzyme cannot be expressed in active form in E. coli . Several factors may account for this, the best studied of which is a binding protein that interacts with large subunits after they are synthesized but before they are assembled into holoenzyme. Various functions for this binding protein are considered.

Characterization of free subunits of ribulose-1,5-bisphosphate carboxylase

Abstract Free subunits of ribulose-1,5-bisphosphate carboxylase (RubPcase) labeled in vivo, and separated from the holoenzyme on sucrose gradients, have been characterized as follows: [ 35 S]methionine label in each of the free subunit pools is chased slowly by either the endogenous or exogenously supplied amino acid. The small subunit pool consists of monomers, while the large subunit pool consists largely of dimers. It is possible that these subunits, or trace amounts of other aggregates, may be intermediates in RubPcase assembly.

Binding of a Transition State Analog to Newly Synthesized Rubisco

Radioactive amino acids, when added to isolated pea chloroplasts or chloroplast extracts engaged in protein synthesis, are incorporated into Rubisco large subunits that co-migrate with native Rubisco during nondenaturing electrophoresis. We have added the transition state analog 2′-carboxyarabinitol bisphosphate (CABP) to chloroplast extracts after in organello or in vitro incorporation of radioactive amino acids into Rubisco large subunits. Upon addition of CABP the radioactive bands co-migrating with native Rubisco undergo a readily detected shift in electrophoretic mobility just as the native enzyme, thus demonstrating the ability of the newly assembled molecules to interact with this transition state analog.

Rubisco Assembly: A Research Memoir

Rubisco is responsible for net carbon dioxide fixation. Due to the high concentration of oxygen in the atmosphere and the relatively low concentration of carbon dioxide, Rubisco “misfires” frequently, splitting a molecule of ribulose bisphosphate rather than adding carbon to it. Evolution has worked to minimize this tendency, but the strategies have been varied, from slight changes in kinetic properties to wholesale re-organization of leaf anatomy. Rubisco consists of two types of subunits in higher plants, green algae, and certain cyanobacteria. The large (L) subunit is encoded in chloroplast DNA and the small (S) subunit in the nucleus. The discovery that Rubisco is encoded by genes in both the chloroplast and the nucleus of higher plants and green algae has motivated considerable research on the biogenesis and biochemistry of Rubisco. This article describes the role of my laboratory in the study of the assembly mechanism of this important enzyme in higher plants.

Computer-assisted simulations of phosphofructokinase-1 kinetics using simplified velocity equations

Equations useful for simulating the kinetic behavior of phosphofructokinase are presented. The equations, which are based on the concerted transition (symmetry) model for allosteric enzymes, account for substrate inhibition by MgATP, cooperative binding by F‐6‐P, activation by F‐2, 6‐P2, and deinhibition by AMP. Velocity calculations can be performed using either a spreadsheet program (e.g., MS Excel) or a web‐based program (e.g., Authorware). Both approaches are illustrated.