FACILITATION OF THE TERMINAL PROTON-TRANSFER REACTION OF RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE BY ACTIVE-SITE LYS166

The terminal step in the carboxylation pathway catalyzed by ribulose 1 ,5-bisphosphate carboxylase/oxygenase (Rubisco) is stereospecific prot onation of the C-2 aci-acid of 3-phosphoglycerate (PGA), X-ray crystal lographic results favor the E-amino group of Lys166 as the proton dono r in this step [Knight et al. (1990) J. Mol. Biol. 215, 113]. Nonethel ess, position-166 mutants are able to catalyze forward processing of i solated 2-carboxy-3-ketoarabinitol 1,5-bisphosphate (CKABP), the carbo xylated reaction intermediate [Lorimer, G. H., & Hartman, F. C. (1988) J. Biol. Chem. 263, 6468]. Prior assays for intermediate processing r elied solely on formation of acid-stable radioactivity from acid-labil e [2'-C-14]CKABP. Therefore, PGA, the normal reaction product, may not have been distinguished from pyruvate, the product from p-elimination of phosphate from the terminal aci-acid intermediate [Andrews, T. J., & Kane, H. J. (1991) J. Biol, Chem. 266, 9447]. If Lys166 indeed serv es as the terminal proton donor, mutants lacking an ionizable side cha in at position 166 might process the carboxylated intermediate predomi nantly to pyruvate. We have thus used anion exchange chromatography an d enzyme coupling to separate and identify the products from turnover of [2'-C-14]CKABP by wild-type, K166G, and K166S enzymes. Although PGA is the only labeled product of significance formed by wild-type enzym e, pyruvate is a major labeled product formed by the mutants. These re sults provide the first direct functionally-based evidence that Lys166 is crucial to the last step in Rubisco-catalyzed conversion of RuBP t o PGA.