Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis Is Caused by Accumulation of a Slow, Tight-Binding Inhibitor at the Catalytic Site.

The slow inactivation which accompanies catalysis by higher-plant ribulose-P(2) carboxylase-oxygenase (Rubisco) in vitro was only partially reversed when the enzyme was gel filtered to remove small molecules. However, gel filtration or dialysis in the presence of high SO(2-) (4) concentrations induced full recovery. This suggests that the inactivation is caused by a tight-binding inhibitor whose effective affinity is reduced by competition with SO(2-) (4) ions, which are known to bind at the catalytic site. The involvement of an inhibitor was confirmed by observations that supernatants obtained after acid-precipitation of inactivated Rubisco were inhibitory when applied to fresh enzyme. The inhibitor bound slowly and tightly and showed strong negative cooperativity. The inhibitor was moderately unstable at pH 8.3, decaying with a halflife of several hours, but was more stable at pH 2. It was destroyed by phosphatase treatment but not by H(2)O(2) or o-phenylenediamine, compounds which react with vicinal dicarbonyl groups. It did not contain a carbon atom derived from substrate CO(2). Possibilities concerning the identity, genesis, and physiological relevance of this inhibitor are discussed.