MUTATIONS IN THE SMALL-SUBUNIT OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE INCREASE THE FORMATION OF THE MISFIRE PRODUCT XYLULOSE-1,5-BISPHOSPHATE/

The small subunit (S) increases the catalytic efficiency of ribulose-1 ,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) by stabilizing the active sites generated by four large subunit (L) dimers. This stabili zation appears to be due to an influence of S on the reaction intermed iate 2,3-enediol, which is formed after the abstraction of a proton fr om the substrate ribulose-1,5-bisphosphate. We tested the functional s ignificance of residues that are conserved among most species in the c arboxy-terminal part of S and analyzed their influence on the kinetic parameters of Synechococcus holoenzymes. The replacements in S (F92S, Q99G, and P108L) resulted in catalytic activities ranging from 95 to 4 3% of wild type. The specificity factors for the three mutant enzymes were little affected (90-96% of wild type), but K-m(CO,) values increa sed 0.5- to 2-fold. Mutant enzymes with replacements Q99G and P108L sh owed increased mis-protonation, relative to carboxylation, of the 2,3- enediol intermediate, forming 2 to 3 times more xylulose-1,5-bisphosph ate per ribulose-1,5-bisphosphate utilized than wild-type or F92S enzy mes. The results suggest that specific alterations of the L/S interfac es and of the hydrophobic core of S are transmitted to the active site by long-range interactions. S interactions with L may restrict the fl exibility of active-site residues in L.