MUTATIONS OF AN ACTIVE-SITE THREONYL RESIDUE PROMOTE BETA-ELIMINATIONAND OTHER SIDE REACTIONS OF THE ENEDIOL INTERMEDIATE OF THE RIBULOSEBISPHOSPHATE CARBOXYLASE REACTION

The side chain of residue threonine 65 within the active site of ribul osebisphosphate carboxylase participates in a network of hydrogen bond s and ionic interactions involving the phosphate moiety attached to C- 1 of the substrate. This residue was replaced with serine, alanine, an d valine in the enzyme from Synechococcus PCC 6301. The mutant enzymes were stable, expressed abundantly by Escherichia coli, and retained t he ability to form gel-filterable complexes with the reaction-intermed iate analog, 2'-carboxyarabinitol-1,5-bisphosphate. The substitutions reduced the K-cat/K-m(CO2) (where K-cat is the substrate-saturated tur nover rate) of the enzyme from 17- to 340-fold with the more radical s ubstitutions causing more severe reductions. The CO2/O-2 specificity a lso deteriorated progressively, the valine replacement causing a 2.3 f old reduction. In concert with these changes, a compound tentatively i dentified as 1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate, the produ ct of beta elimination of the 2,3-enediol(ate) intermediate of the cat alytic reaction, appeared among the reaction products in progressively increasing amounts. In the case of the valine substitution, it compri sed 13% of the ribulose bisphosphate consumed. The mutant enzymes also partitioned more of their reaction flux to pentulose bisphosphate iso mers of ribulose bisphosphate. By contrast, the diversion of carboxyla ted product to pyruvate, as a result of beta elimination of the three- carbon aci-carbanion intermediate of the carboxylation reaction, was a meliorated by the replacements, the valine mutant showing a 5-fold imp rovement in this parameter. These observations focus attention on a ge ometric conflict which exists between the requirements for stabilizati on of the 5-carbon enediol(ate) and 3-carbon aci-carbanion intermediat es. This conflict must be resolved by a change in the angle of the C-1 /bridge oxygen bond during each catalytic cycle. The network of hydrog en bonds involving the side chain of threonine 65 must play a crucial role in facilitating reaction of the enediol(ate) with the gaseous sub strate and in shepherding this subsequent movement.