BETA-ELIMINATION OF PHOSPHATE FROM REACTION INTERMEDIATES BY SITE-DIRECTED MUTANTS OF RIBULOSE-BISPHOSPHATE CARBOXYLASE OXYGENASE

Five residues (Thr-53, Asn-54, Gly-370, Gly-393, and Gly-394) of Rhodo spirillum rubrum ribulose-bisphosphate carboxylase/oxygenase are posit ioned to serve as hydrogen-bond donors for the C1 phosphate of ribulos e bisphosphate and thereby constrain conformational flexibility of the initial enediol(ate) intermediate (Knight, S., Andersson, I., and Bra nden, C.-I. (1990) J. Mol. Biol. 215, 113-160). To study the functiona l contributions of the residues implicated in ribulose bisphosphate bi nding and intermediate stabilization, we have replaced them individual ly with alanine, either to remove the H-bonding group (T53A, N54A) or to introduce bulk (G370A, G393A, G394A). Consequences of substitutions include diminution of carboxylase activity (with a lesser impact on e nolization activity), increase of K(m) (ribulose bisphosphate), and de crease of carboxylation: oxygenation specificity. During catalytic tur nover of ribulose bisphosphate by several mutants, substantial amounts of the substrate are diverted to 1-deoxy-D-glycero-2,3-pentodiulose 5 -phosphate, reflecting beta-elimination of phosphate from the enediol( ate) intermediate. This side product is not observed with wild-type en zyme, nor has it been reported with mutant enzymes characterized previ ously. Another consequence of disruption of the phosphate binding site is enhanced production of pyruvate, relative to wild-type enzyme, by some of the mutants due to decomposition of the aci-carbanion of 3-pho sphoglycerate (the terminal intermediate). These data provide direct e vidence that phosphate ligands stabilize conformations of intermediate s that favor productive turnover and mitigate beta-elimination at two stages of overall catalysis.