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Earlier structural and functional studies of ribulose-1,5-bisphosphate carboxylase/oxygenase imply that K334 facilitates the addition of gaseous substrate to the 2,3-enediol(ate) derived from ribulose 1,5-bisphosphate. Crystallographic analysis of the activated spinach enzyme [Knight et al. (1990) J. Mol. Biol. 215, 113-160] shows that the lysyl side chain is appropriately positioned to stabilize the transition state for the addition of CO2 to the enediol(ate). Furthermore, despite total impairment of carboxylase and oxygenase activities, site-directed mutants of the Rhodospirillum rubrum enzyme with replacements for lysine K334 (formerly designated K329) retain the capacity to enolize ribulose 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 that the K334C mutant is also competent in the latter stages of catalysis, whereby 2'-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon intermediate of the carboxylation pathway, is correctly processed to 3-phosphoglycerate. Thus, the impairment of the mutant in overall catalysis can be attributed to preferential disruption of the reaction of CO2 or O2 with the enzyme-bound enediol(ate). Chemical rescue of the K334C mutant by aminoethylation and aminopropylation shows that this disruption reflects, at least in part, a failure to adequately stabilize the relevant transition state. With several simplifying assumptions, the CO2/O2 specificity factor tau can be reduced to the ratio of the fundamental second-order rate constants for the interaction of the gaseous substrates with the enzyme-bound 2,3-enediol(ate) of ribulose bisphosphate.(ABSTRACT TRUNCATED AT 250 WORDS)