Anthony A. Gatenby

Rubisco Synthesis, Assembly, Mechanism, and Regulation.

abstractlon - H cls -enedlol o=o ribulose 1,5-P, / OPO, + 2P glycolate OPO, 3P Dglycerate OPO, 3-keto arabinitol 1 ,5-P2 Figure 4. The Reactions Catalyzed by Rubisco. The first intermediate of catalysis is the C2,C3 cis-enediol form of ribulose-bisphosphate (ribulose 1,5-P2) after abstraction of the C3 proton. The enediol can partition a number of ways, the majority into the products of carboxylation (upper reactions) or oxygenation (lwer reactions). However, a number of misprotonated isomers of ribulose-bisphosphate, for example, xylulose-bisphosphate, have been detected with the wild-type enzyme that are produced in quantity by mutations of specific amino acids involved in proton transfer. Phosphate elimination of the carbanion forms of intermediates are also produced by some mutants (see Morrell et al., 1994). R, -CHOH-CH20P03= ; 3P D-glycerate, 3-phosphoglycerate; 2P glyco- late, 2-phosphoglycolate. to bind. Coordination of the metal does not complete the ac- tive site but simply positions the carbamate and Mg2+ relative to those centers of the bisphosphate substrate, namely, the C2 carbonyl oxygen and C3 hydroxyl, that are involved in the catalytic events. With substrate bound in the correct orienta- tion (Lorimer et al., 1989), the C2 and C3 oxygen atoms complete the coordination sphere (Gutteridge and Lundqvist, 1994). The catalytic mechanism of the carboxylation of ribulose- bisphosphate can be depicted as five discrete partia1 reactions (Andrews and Lorimer, 1987). The first step has been estab- lished as the formation of an enediol intermediate of the bisphosphate substrate that is catalyzed by the enzyme in the absence of the second substrates,

Assembly of cyanobacterial and higher plant ribulose bisphosphate carboxylase subunits into functional homologous and heterologous enzyme molecules in Escherichia coli

The genes for the large (rbcL) and small (rbcS) subunits of ribulose‐1,5 bisphosphate carboxylase‐oxygenase (RuBPCase) from the cyanobacterium Synechococcus PCC 6301, and the rbcS gene of wheat, have been expressed in Escherichia coli in order to study homologous and heterologous enzyme assembly. Synechococcus L subunits expressed in E. coli in the absence of S subunits assemble into oligomeric structures without detectable enzyme activity. Co‐expression of L and S subunits, achieved after infection with an M13 recombinant phage containing the rbcS gene, restores enzyme activity, thus demonstrating the essential role of S in the formation of an active RuBPCase. The S subunit, however, is neither required for the solubility nor for the assembly of the L subunits into oligomeric forms. The specific activity of the homologous Synechococcus RuBPCase can be modulated by changing the intracellular pool size of S by phage infection. Heterologous assembly between L subunits of Synechococcus and S subunits of wheat can be demonstrated and results in a functional enzyme. The hybrid RuBPCase has approximately 10% of the activity of the homologous Synechococcus enzyme.

3 – Chaperonins of Photosynthetic Organisms

Publisher Summary A number of the features more recently described for bacterial and mitochondrial chaperonins that can be found in the early literature on chloroplast chaperonins, such as binding of nascent polypeptides to the chaperonin oligomer and the requirement for MgATP to release the bound target polypeptide. Because these studies described the binding of the large (L) subunit of ribulose-bisphosphate carboxylase-oxygenase to an oligomeric “assembly” protein, this oligomer was termed the large subunit binding protein (LSBP). The name was later modified to the Rubisco subunit binding protein when it was realized that the small (S) subunit of Rubisco could also bind to the LSBP. The complex molecular architecture of chaperonins clearly has important mechanistic significance, because the pattern of seven-fold rotational symmetry is repeated for cpn60 homologs isolated from chloroplasts and mitochondria. Members of a second chaperonin subfamily that functionally interact with cpn60 are also oligomeric proteins, but are significantly smaller than cpn60, and usually contain identical subunits of about 10 kDa. This smaller chaperonin protein is known as GroES in E. coli and is more generally referred to as chaperonin 10 (cpn 10), reflecting its subunit size.