Protein liganding to the activator cation of ribulose bisphosphate carboxylase

Abstract

Spinach leaf ribulosebisphosphate carboxylase forms a quaternary complex with CO2, carboxyarabinitol bisphosphate, and Cr2+ or Co2+. Oxidation of the cation in these complexes produces a protein--cation adduct which is sufficiently stable to be chromatographically isolated after enzyme denaturation. While stoichiometric levels of slowly exchanging cation can be specifically trapped after addition of protein denaturants as well as a vast molar excess of Mg2+, neither CO2 nor carboxyarabinitol bisphosphate remains bound to denatured protein under the conditions employed in these experiments. These observations demonstrate direct innersphere liganding of protein to the exchange-inert cation, which appears to bind at the site normally occupied by the physiologically active cation. Dimeric ribulosebisphosphate carboxylase from Rhodospirillum rubrum also forms a quaternary complex containing stoichiometric amounts of enzyme protomer, CO2, Co2+, and carboxyarabinitol bisphosphate. Lack of a small subunit in the R. rubrum enzyme does not impair binding of the components of the quaternary complex in a nonexchangeable mode. Substantial amounts of protein--cation adduct are recovered upon oxidation and denaturation of the R. rubrum complex, supporting the prediction that the large subunits of the octameric plant enzyme should be the sites of cation binding. The first direct proof for such a hypothesis has been generated by separation of protein subunits derived from a spinach quaternary complex and by the demonstration that the bound cation is associated with the large subunit.

Ribulose-1,5-Bisphosphate Carboxylase-Oxygenase

“…Stereochemical constraints involved in the mechanism of carboxylation have been proposed on the basis of analog experiments (156) and the existence of a formally hydrated gem-diol form of the 2-carboxy-3-keto arabinitol bisphosphate reaction intermediate has been predicted (154) on the basis of studies on the slow, tight-binding inhibitors. In addition, model complexes have clearly indicated that only one cation is bound to each enzyme site that is fully occupied with activator CO2 as well as substrate CO2 and RuBP; this observation applies to the dimeric R. rub rum enzyme as well as the octameric plant enzymes (83) and sets some constraints on the possible roles for cation and on the interaction between sites of activation and catalysis (121).…”

“…If cation supports activation by coordinating directly to a carbamate and functions in catalysis while binding in close proximity to substrate CO 2 , then, since only one cation binds per active site (83,117,121), the sites for activation and catalysis are in close proximity. With the primary sequence of the large subunit known (58), and several peptides involved in activation (65) and catalysis (cf below) sequenced and positioned within the large subunit backbone, a correct juxtaposition of these regions may expedite the long-range process of generating a three dimensional model of the enzyme.…”

A site-specific mutation within the active site of ribulose-1,5-bisphosphate carboxylase of Rhodospirillum rubrum

“…and Reddy, 1984). The divalent metal ion influences the partitioning between carboxylation and oxygenation (Wildner and Henkel, 1979;Christeller, 1981;Jordan and Ogren, 1983) suggesting that, besides its involvement in activation, it is important in catalysis (Miziorko and Sealy, 1980;Miziorko et al, 1982;Pierce et al, 1980). The stoichiometry (Miziorko and Sealy, 1980;Miziorko et al, 1982) (1:1:1:1) of the quaternary complex enzyme.activator carbamate.Mn2 +.2-carboxyarabinitol 1,5-bisphosphate, containing the tightly-bound, reactionintermediate analog, 2-carboxyarabinitol 1,5-bisphosphate (2-CABP) (Pierce et al, 1980;Schloss and Lorimer, 1982), also implies the same dual role.…”

Formation and Characterization of an Active Phosphoenolpyruvate Carboxykinase−Cobalt(III) Complex

“…Balakrishnan and Villafranca (1979) reported the preparation of both the Co 3+ and Cr 3+ glutamine synthetase derivatives. Miziorko et al (1982) reported the stoichiometric incorporation of Co 3+ into ribulosebisphosphate carboxylase. The Co 3+ -modified ribulosebisphosphate carboxylase was inactive but still permitted the binding of substrates.…”