With the mass convergent evolution of the C-fixation pathway in a diversity of plant lineages, ancestral C-type RuBisCO evolved to have faster turnover of  in exchange for lower specificity as a result of the greater localization of  from the mesophyll cells into the bundle sheath cells. This was achieved through enhancement of conformational flexibility of the “open-closed” transition in the Calvin cycle. Laboratory-based phylogenetic studies have shown that this evolution was constrained by the trade-off between stability and activity brought about by the series of necessary mutations for C RuBisCO. Moreover, in order to sustain the destabilizing mutations, the evolution to C RuBisCO was preceded by a period in which mutations granted the enzyme increased stability, establishing a buffer to sustain and maintain the mutations required for C RuBisCO. To assist with this buffering process, the newly-evolved enzyme was found to have further developed a series of stabilizing mutations. While RuBisCO has always been accumulating new mutations, most of these mutations that have survived have not had significant effects on protein stability. The destabilizing C mutations on RuBisCO has been sustained by environmental pressures such as low  concentrations, requiring a sacrifice of stability for new adaptive functions.