The stereochemical course of ribulosebisphosphate carboxylase. Reductive trapping of the 6-carbon reaction-intermediate

Abstract

The carboxylation of ribulosebisphosphate involves a 6-carbon reaction-intermediate, 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate. This labile intermediate, radiolabeled with 14CO2, was reduced (and thereby stabilized) with NaB[3H4]. This yielded a doubly labeled mixture with the chromatographic properties of 2-carboxy- and 4-carboxy-D-arabinitol 1,5-bisphosphate. These products (as their respective gamma-lactones) were chromatographically resolved from the gamma-lactones of 2-carboxy-D-ribitol and 2-carboxy-D-xylitol 1,5-bisphosphate, the other possible reduction products to be considered. Reduction of the 6-carbon reaction-intermediate by NaBH4 required prior denaturation of the enzyme. Of various denaturants tested, only acid was rapid enough to give good yields of the carboxypentitol bisphosphates. The reduction products of the reaction-intermediate inhibited the carboxylase stoichiometrically. In contrast to 2-carboxyribitol and 2-carboxyxylitol bisphosphate, 2-carboxy- and 4-carboxyarabinitol bisphosphate are exceptionally potent, essentially irreversible, slow-binding inhibitors of ribulosebisphosphate carboxylase.