Structural and evolutionary factors affecting the tight binding of inhibitors by ribulose 1,5-bisphosphate carboxylase/oxygenase.

Abstract

The large (L) and small (S) subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) from Synechococcus PCC 6301 were expressed separately and a method was developed for their purification from Escherichia coli. The Synechococcus L subunits could be assembled with either Synechococcus S or pea S and the respective holoenzymes had high specific activities. Using a method developed for determination of carbamylation of L, the pK's for activation were found to be lower for L₈S₈ relative to L while the K(act)'s for Co₂ were not different. Purified L was analyzed by gel filtration and eluted at a position consistent with it being octameric. The transition state analog, 2-carboxy- scD-arabinitol 1,5-bisphosphate (CABP) bound tightly to L₈. Incubation at 0.6 M ionic strength increased the dissociation rate constant for the L-CABP complex by three orders of magnitude over that observed for L₈S₈. The binding of CABP to L caused a major change in the gel filtration profile implying that a conformational change had occurred. The binding of 2-carboxyribitol 1,5-bisphosphate (CRBP) had no such effect, indicating that the shift was due to the tight binding of CABP. At 0.6 M ionic strength in the presence of CABP or in the presence of S and CABP, the change was not observed. These results indicate that alterations in interactions at the L-S interface participate in CABP tight binding. Synechococcus rubisco had higher overall K(d)'s relative to spinach rubisco for 2-carboxy- scD-arabinitol 1-phosphate and xylulose 1,5-bisphosphate. The Kᵢ's for these inhibitors with respect to RuBP were similar, indicating that the difference in overall affinity was due to the conformational changes involved in tight binding. RuBP inhibited the rate of activation of a Synechococcus L₈-pea S hybrid to a greater extent than the native Synechococcus L₈S₈ holoenzyme. Synechococcus L₈ was activated by RuBP indicating that S influences the affinity of RuBP for the inactive form. Rubisco activase from spinach was unable to activate RuBP inhibited Synechococcus, or a Synechococcus L₈-pea S hybrid enzyme. Rubiscos from higher plants have a greater ability to discriminate between CO₂ and O₂ as substrates than do rubiscos from cyanobacteria. This characteristic was probably required for survival in high O₂, low CO₂ atmospheres. Inhibitor tight binding may have been an undesired side effect of the evolution of increased specificity for CO₂. Rubisco activase may have evolved simultaneously with specificity factor to allow higher plant rubiscos to function in the presence of inhibitors.