Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis Is Not Due to Decarbamylation of the Catalytic Site

Abstract

An investigation was made of the proposal that the slow inactivation of ribulosebisphosphate carboxylase (Rubisco) activity, which occurs during in vitro assays, is due to decarbamylation of the enzyme. The level of carbamylation was compared with catalytic activity during assay conditions in which activity was both increasing and decreasing. Carbamylation level was measured using the reaction-intermediate analogue 2'-carboxy-D-arabinitol-1,5-bisphosphate (carboxyarabinitol-P2). A dual isotope procedure was used in which [3H]carboxyarabinitol-P2 measured total active sites and 14CO2 reported the level of carbamylation.The efficacy of the procedure was verified both in the presence and in the absence of the substrate D-ribulose-1,5-bisphosphate (ribulose-P2). These measurements showed that changes in activity during assays were not correlated with carbamylation status. Inactivation during assays initiated with both fully and partially carbamylated enzyme was not associated with any change in carbamylation level. This implies that the loss of activity during assays is not due to ribulose-P2 binding and sequestering the E form of the enzyme. Ribulose-P2 did not appear to alter the equilibrium between carbamylated and uncarbamylated enzyme, but it did slow the rate at which enzyme was both decarbamylated and carbamylated. The most likely explanation for the loss of activity during assays appears to be the sequestration of carbamylated, Mg2 -bound active sites by an inhibitor. (ECM). This theory has been based on experimental findings which show that phosphorylated sugars, including ribulose-P2, may bind to both the E and ECM forms of the enzyme at the catalytic site (2, 14). When bound to the ECM form, the activating CO2 and Mg2' are stabilized on the enzyme. When bound to the E form, carbamylation is retarded. Depending on the relative affinity ofthese compounds for E and ECM, they may shift the equilibrium between these forms of the enzyme.There have been several indications that ribulose-P2 binds tightly to the E form of Rubisco. For example, Jordan and Chollet (9) measured a dissociation constant for the E-ribulose-P2 complex of 21 nM at 2°C, and Laing and Christeller ( 11) showed that activation of E was very slow in the presence of ribulose-P2. With such tight binding of ribulose-P2 to E, it could readily be expected that there should be a decline in the level of ECM during carboxylation, and conversely, that there should be very little increase in the level of ECM when E is added to ribulose-P2 in the presence of CO2 and Mg2'. In fact, it is thought that the enzyme, Rubisco activase, is required in vivo to promote activation in the presence of ribulose-P2 (20). Contrary to this, there are a number of reports of ribulose-P2 promoting carbamylation of Rubisco. Vater et al. (22) showed that ribulose-P2 induced a decrease in the dissociation constant for the EC complex similar to that seen with sugar phosphate activators such as 6-phosphogluconate, and NADPH (2,5,14). Additionally, Laing and Christelle...

Side Reactions Catalyzed by Ribulose-bisphosphate Carboxylase in the Presence and Absence of Small Subunits

“…Glycerate presumably is formed by dephosphorylation of a portion of the major product P-glycerate by traces of phosphatases contaminating the enzyme preparations. The large enzyme concentrations and extended assay periods necessitated by the feeble activity of L 8 system. The partitioning ratios observed were 0.61 Ϯ 0.05% (n ϭ 6) and 0.71 Ϯ 0.05% (n ϭ 5) for L 8 (Fig.…”

“…The large enzyme concentrations and extended assay periods necessitated by the feeble activity of L 8 system. The partitioning ratios observed were 0.61 Ϯ 0.05% (n ϭ 6) and 0.71 Ϯ 0.05% (n ϭ 5) for L 8 (Fig. 1A) and L 8 S 8 (Fig.…”

“…For the spinach enzyme, this might be a result of inhibition caused by accumulating misprotonation by-products. Indeed, the kinetics of the decline for the spinach enzyme appeared quite reminiscent of the decline usually seen during catalysis which has been attributed to that cause (7)(8)(9)(10). Synechococcus Rubisco, however, is not subject to progressive inactivation during catalysis, at least at CO 2 saturation (16), and it showed a more pronounced decline such that, after 15-20 min, the rate of absorbance increase had fallen to approach the basal rate seen in the enzyme-free control.…”

“…Preparation of L 8 Rubisco-E. coli HB101 harboring plasmid pDB53, which bears the Synechococcus PCC 6301 rbcL gene under the transcriptional control of the lac promoter (28), was used to produce large subunit octamers (L 8 ) devoid of small subunits. L 8 was extracted from E. coli, purified, and stored using the same procedures developed for the Synechococcus L 8 S 8 holoenzyme (16).…”

A Kinetic Characterization of Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis

“…There have been many proposals that ribulose-P2 binds more tightly to the uncarbamylated enzyme (E) than to the carbamylated, metal-complexed form (ECM), thus promoting decarbamylation with concomitant loss of activity (11,12,15). This plausible hypothesis has been tested, and found to be incorrect, in the work described in the second paper ofthis series (8). (b) Readily reversible binding of ribulose-P2 to a regulatory site, different from the catalytic site, causing a reduction in activity of the fully carbamylated enzyme has been proposed by Yokota and Kitaoka (26) who suggested that fallover is a natural phenomenon, reflecting in vivo control of Rubisco activity by ribulose-P2.…”