PARENT SESSION
Posters P3C C3 and Rubisco. Abstracts (617-630)

Site-directed mutagenesis of tyrosine residues from beta A/B loop Rubisco small-subunit induces a divergent proteolytic fragmentation pattern of large-subunit. Maria Gloria Esquivel^*,, Joaquin Moreno,

ABSTRACT- Ribulose-1,5- bisphosphate carboxylase/oxygenase (rubisco) catalyzes the CO2 fixation step in biological carbon assimilation. The chloroplastic holoenzyme has 8 large subunits, containing the active site, and 8 small subunits. The role of the small subunit remains unclear. However, it has been demonstrated that the loop between beta strands A and B of the small subunit can influence catalytic efficiency, CO2/O2 specificity, and holoenzyme stability. On the other hand, it has been recently shown that the proteolytic fragmentation pattern of the large subunit of rubisco depends on the redox state of critical cysteines, and that the kinetic analysis of Rubisco proteolysis in the reduced and oxidized state may be used to detect subtle changes of enzyme conformation. A possible contribution of the small subunit to the proteolytic susceptibility of rubisco remains to be examined. We have studied the proteolytic fragmentation of the large subunit of rubisco in 3 different tyrosine mutants from the loop between beta strands A and B of rubisco small-subunit, Y67A, Y68A e Y72A. The mutants were created by site-directed mutagenesis and transformation of a C. reinhardtii strain that lacks the small subnit gene family. Purified rubisco, from those mutants and from wild type revertants, was oxidized (treated with the disulfide cystamine) or reduced (treated with the free thiol cysteamine) before being incubated with subtilisin for fixed times. The large subunit fragmentation was investigated by SDS-PAGE. Analysis of the gels indicated a significant alteration of the proteolytic fragmentation pattern of the large subunit in all mutant enzymes. The Y67A and Y72A mutant rubiscos were more sensitive to proteolysis than Y68A, and their oxidized forms were directly degraded to low molecular products, without detectable fragments. These results suggest that certain residues from the small subunit play an important role on the structural conformation of the large subunit and on the proteolytic susceptibility of the rubisco holoenzyme.

KEY WORDS: proteolysis, chloroplast, Rubisco, Chlamydomonas reinhardtii