Rubisco activase

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. 

As discussed in Section 22.7, illumination of chloroplasts leads to light-driven pumping of protons into the thylakoid lumen, which causes pH changes in both the stroma and the thylakoid lumen (Figure 22.27). The stromal pH rises, typically to pH 8. Because rubisco and rubisco activase are more active at pH 8, COg fixation is activated as stromal pH rises. Fructose-1,6-bisphosphatase, ribulose-5-phosphate kinase, and glyceraldehyde-3-phosphate dehydrogenase all have alkaline pH optima. Thus, their activities increase as a result of the light-induced pH increase in the stroma. 

Another regulatory mechanism involves the nocturnal inhibitor 2-carboxyarabinitol 1-phosphate, a naturally occurring transition-state analog (see Box 6-3) with a structure similar to that of the /3-keto acid intermediate of the rubisco reaction (Fig. 20-7 see also Fig. 20-20). This compound, synthesized in the dark in some plants, is a potent inhibitor of carbamoylated ru-bisco. It is either broken down when light returns or is expelled by rubisco activase, activating the rubisco. 

Rubisco condenses C02 with ribulose 1,5-bisphosphate, forming an unstable hexose bisphosphate that splits into two molecules of 3-phosphoglycerate. Rubisco is activated by covalent modification (carbamoylation of Lys201) catalyzed by rubisco activase and is inhibited by a natural transition-state analog, whose concentration rises in the dark and falls during daylight. 

Calvin cycle 752 plastids 752 chloroplast 752 amyloplast 752 carbon-fixation reaction 753 ribulose 1,5-bisphosphate 753 3-phosphoglycerate 753 pentose phosphate pathway 753 reductive pentose phosphate pathway 753 C3 plants 754 ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) 754 rubisco activase 757... 

Portis, A.R., Jr. (2003) Rubisco activase rubisco s catalytic chaperon. Photosynth. Res. 75, 11-27. 

Structure, regulation, mechanism, and importance of rubisco activase. 

A. Rokka, L. Zhang, and E.-M. Aro. 2001. Rubisco activase An enzyme with a temperature-dependent dual function Plant J. 25 463-472. (PubMed)... 

N. Zhang and A.R. Portis Jr. 1999. Mechanism of light regulation of rubisco A specific role for the larger rubisco activase isoform involving reductive activation by thioredoxin-f Proc. Natl. Acad. Sci. USA 96 9438-9443. (PubMed) (Full Text in PMC)... 

There are many other phenomena specifically observed in H-COj conditions. For example, H-COj cells of C. littorale exhibit higher Rubisco activity than L-COj cells, due to the increase in the amount of this enzyme detected by antibody [11]. Rubisco activase is also higher, but phosphoenolpyruvate carboxylase activity is lower in H-... 

FIGURE 20-8 Roie of rubisco activase in the carbamoyiation of of rubisco. When the substrate ribulose 1,5-bisphosphate is bound to the active site, Lys ° is not accessible. Rubisco activase couples ATP hydrolysis to expulsion of the bound sugar bisphosphate, exposing Lys this Lys residue can now be carbamoylated with CO2 in a reaction that is apparently not enzyme-mediated. is attracted to... 

REGULATION OF RIBULOSE BISPHOSPHATE CARBOXYLASE ACTIVITY BY RUBISCO ACTIVASE ASPECTS OF THE MECHANISM... 

The rca mutant lacks two nearly identical polypeptides of about 45 and 41 kD (2,6). The ability to observe a rubisco activase dependent activation of rubisco in vitro was followed by the rapid development of procedures to purify the protein for detailed biochemical study (7) and the cloning and sequencing of the rubisco activase gene (8). 

It is clear that rubisco activase is an essential and probably the primary factor in the regulation of rubisco activity. Now a detailed understanding of the mechanism of activation by the protein is of major concern in order to obtain a complete understanding of how rubisco activity may be regulated in vivo. Some basic clues as to how rubisco activase may be functioning to alter rubisco activity have been obtained by investigating various features of the process in vitro. 

The presence of RuBP makes the rapid activation of rubisco dependent on the presence of rubisco activase (Fig 1). The addition of RuBP to the inactive form of the enzyme before CO2 and Mg are added makes activation extremely slow. In contrast, the presence of rubisco activase counteracts the inhibitory nature of RuBP allowing activation to occur rapidly. The active enzyme obtained in this manner appears to the same as that generated by the spontaneous process in the absence of... 

FIGURE 1. Activation of rubisco (100 fig/ml) in the presence and absence of rubisco activase (270 /ig/ml) and/or RuBP (6mM) at high and low CO2. An ATP regenerating system using creatine phospho-kinase was also present. (A.R. Portis, unpublished)... 

In this assay system, an ATP/ADP ratio of only 10 1 is sufficient to inhibit rubisco activase activity by 50% but in the isolated chloroplast studies rubisco activation is reduced by 50% at ATP/ADP ratios of about 1 1. These values are not directly comparable however, because all of the factors affecting the relationship between rubisco activase activity and the resulting steady state rubisco activity are not yet clear. 

FIGURE 2. Effects of ATP and ADP concentration on rubisco activase dependent activation of rubisco (from ref. 14). 

The ATP requirement for rubisco activase activity led to the finding that rubisco activase has an intrinsic ATP hydrolysis activity (14). The concentration dependence on ATP of the rubisco activation and ATP hydrolysis activities are identical (Fig. 3) and equally sensitive to ADP (not shown). However, the ATP hydrolysis activity is not strictly coupled to the activation of rubisco (Table I) and proceeds regardless of the presence of rubisco in various forms. On the other hand a loss of the ATPase activity by heating or various chemical inhibitors also causes a corresponding loss activation activity (14). Recently we have been able to reversibly alter the ATPase activity of the isolated protein and also change its apparent size on gel permeation columns by various treatments (Z.Y. Wang and... 

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