The activation of ribulose-1,5-bisphosphate carboxylase by carbon dioxide and magnesium ions. Equilibria, kinetics, a suggested mechanism, and physiological implications

Abstract

Ribulose-1,5-bisphosphate carboxylase was activated by incubation with CO2 and Mg2++, and inactivated upon removal of CO2 and Mg2+ by gel filtration. The activation process involved CO2 rather than HCO3-. The activity of the enzyme was dependent upon the preincubation concentrations of CO2 and Mg2+ and upon the preincubation pH, indicating that activation involved the reversible formation of an equilibrium complex of enzyme-CO2-Mg. The initial rate of activation was linearly dependent upon the CO2 concentration but independent of the Mg2+ concentration. Kinetic analyses indicated that the enzyme reacted first with CO2 in a rate-determining and reversible step, followed by a rapid reaction with Mg2+ to form an active ternary complex (see eq 1 in text). The pseudo-first order rate constant, kobsd, for the activation process at constant pH was derived: kobsd=k1[CO2] + (k2k4/k3[Mg2+]). Experimentally, kobsd was shown to be linearly dependent upon the CO2 concentration and inversely dependent upon the Mg2+ concentration. The activity of the enzyme after preincubation to equilibrium at constant concentrations of CO2 and Mg2+ increased as the preincubation pH was raised, indicating that CO2 reacted with an enzyme group whose pK was distinctly alkaline. It is proposed that the activation of ribulose-1, 5-biphosphate carboxylane involves the formation of a carbamate.

Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5‐bisphosphate carboxylase/oxygenase

“…Reflective of the high affinity of activated Rubisco for CABP (Pierce et ai., 1980), formation of a stable, stoichiometric complex between the enzyme and the reaction-intermediate analogue is diagnostic of proper activation [carbamylation of active-site Lys 191 and binding of Mg2' (Lorimer et al, 1976;Lorimer, 1981;Donnelly et al, 1983)l. H287N and H287Q both form Mg2+-dependent complexes containing -1 molecule of CABP per active site, as reported previously for the H287N mutant (Lorimer et ai., 1987;Gutteridge et al, 1988).…”

Activation of Ribulosebisphosphate Carboxylase/Oxygenase at Physiological CO2 and Ribulosebisphosphate Concentrations by Rubisco Activase

“…The K,,t(CO2) of rubisco activation in the reconstituted system was 4 micromolar C02, compared to a K,A,(C02) of 25 to 30 micromolar CO2 for the previously reported spontaneous CO2/Mg2' activation mechanism. The activation process characterized here explains the high degree of rubisco activation at the physiological concentrations of 10 micromolar CO2 and 2 to 4 millimolar RuBP found in intact leaves, conditions which lead to almost complete deactivation of rubisco in vitro.The photosynthetic assimilation ofCO2 is initiated by rubisco.2 This enzyme exhibits complex regulatory properties, including a requirement that it be converted to an activated state in order to acquire catalytic competency (8,9,14). The activation of rubisco in vitro occurs by the spontaneous addition of activating CO2 (ACO2) to the e-amino group of a lysine residue near the active site, followed by the addition of Mg2' (Eq.…”

Formation of stoichiometric cobalt(III)/CO2 complexes with spinach ribulose bisphosphate carboxylase/oxygenase

“…The enzyme may also act as an internal monooxygenase and catalyze the oxygenolysis of RuBP, leading to the production of glycolic acid during photorespiration (reviewed [ 11). For maximum catalytic rates, the enzyme must first be activated by preincubation with a divalent cation and CO2 [2,3] and there is some evidence to suggest that the CO2 activation site is distinct from the catalytic site [4,5]. With regard to the divalent cation requirement of the eucaryotic enzyme, it has been shown that Mn*', Ni*' and Co*+ will substitute for Mg*+ for both carboxylase [6] and oxygenase [7] activities.…”