A model for the kinetics of activation and catalysis of ribulose 1,5-bisphosphate carboxylase

Abstract

Further evidence for time-dependent interconversions between active and inactive states of ribulose 1,5-bisphosphate carboxylase is presented. It was found that ribulose bisphosphate oxygenase and ribulose bisphosphate carboxylase could be totally inactivated by excluding CO2 and Mg2+ during dialysis of the enzyme at 4 degrees C. When initially inactive enzyme was assayed, the rate of reaction continually increased with time, and the rate was inversely related to the ribulose bisphosphare concentration. The initial rate of fully activated enzyme showed normal Michaelis-Menten kinetics with respect to ribulose bisphosphate (Km = 10muM). Activation was shown to depend on both CO2 and Mg2+ concentrations, with equilibrium constants for activation of about 100muM and 1 mM respectively. In contrast with activation, catalysis appeared to be independent of Mg2+ concentration, but dependent on CO2 concentration, with a Km(CO2) of about 10muM. By studying activation and de-activation of ribulose bisphosphate carboxylase as a function of CO2 and Mg2+ concentrations, the values of the kinetic constants for these actions have been determined. We propose a model for activation and catalysis of ribulose bisphosphate carboxylase: (see book) where E represents free inactive enzyme; complex in parentheses, activated enzyme; R, ribulose bisphosphate; M, Mg2+; C, CO2; P, the product. We propose that ribulose bisphosphate can bind to both the active and inactive forms of the enzyme, and slow inter-conversion between the two states occurs.

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

“…This activation process is clearly distinguishable from the spontaneous in vitro activation process characterized previously (8,9) which is severely inhibited by preincubation with RuBP (3,6,8). The rubisco activation system also differs from the chloroplast ferredoxin-thioredoxin system, which catalyzes the activation of several photosynthetic enzymes by reduction with electrons derived from water (2), since rubisco activation proceeds in the presence of the artificial electron transport acceptors methylviologen (17) and pyocyanine.…”

“…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.…”

“…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.…”

The discovery of Rubisco activase — yet another story of serendipity

“…Meanwhile, the other substrate, RuBP, received little notice even though the early paper by Pon et al (1963) also showed that preincubation of the enzyme with RuBP led to reduced rates of activity. Later, a detailed analysis by Laing and Christeller (1976) provided evidence that RuBP was an inhibitor of activation, allowing them to extend the carbamylation model of Lorimer et al (1976). Finally, a paper by Jordan and Chollet (1983) most clearly showed that RuBP binding to the uncarbamylated enzyme resulted in a potent inhibition of the carbamylation/activation process.…”