A ROLE FOR THE EPSILON-AMINO GROUP OF LYSINE-334 OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE IN THE ADDITION OF CARBON-DIOXIDE TO THE 2,3-ENEDIOL(ATE) OF RIBULOSE 1,5-BISPHOSPHATE

Earlier structural and functional studies of ribulose-1,5-bisphosphate carboxylase/oxygenase imply that K334 facilitates the addition of gas eous substrate to the 2,3-enediol(ate) derived from ribulose 1,5-bisph osphate. Crystallographic analysis of the activated spinach enzyme [Kn ight et al. (1990) J. Mol. Biol. 215, 113-160] shows that the lysyl si de chain is appropriately positioned to stabilize the transition state for the addition of CO2 to the enediol(ate). Furthermore, despite tot al impairment of carboxylase and oxygenase activities, site-directed m utants of the Rhodospirillum rubrum enzyme with replacements for lysin e K334 (formerly designated K329) retain the capacity to enolize ribul ose bisphosphate, demonstrating that the primary catalytic lesion lies beyond this initial step [Soper et al. (1988) Protein Eng. 2, 39-44; Hartman & Lee (1989) J. Biol. Chem. 264, 11784-11789]. We now show tha t the K334C mutant is also competent in the latter stages of catalysis , whereby 2'-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-car bon intermediate of the carboxylation pathway, is correctly processed to 3-phosphoglycerate. Thus, the impairment of the mutant in overall c atalysis can be attributed to preferential disruption of the reaction of CO2 or O2 with the enzyme-bound enediol(ate). Chemical rescue of th e K334C mutant by aminoethylation and aminopropylation shows that this disruption reflects, at least in part, a failure to adequately stabil ize the relevant transition state. With several simplifying assumption s, the CO2/O2 specificity factor tau can be reduced to the ratio of th e fundamental second-order rate constants for the interaction of the g aseous substrates with the enzyme-bound 2,3-enediol(ate) of ribulose b isphosphate. The tau-value is therefore a measure of the differential stabilization of the transition states that lead to the carboxylated a nd oxygenated intermediates. Our observations of altered tau-values an d hence differential perturbation of transition states, correlated wit h substitutions of K334 with S-(2-aminoethyl)cysteine and S-(3-aminopr opyl)cysteine, lend strong credence to one assigned role for this lysy l residue.