Article

Structural and functional properties of a multi-enzyme complex from spinach chloroplasts. 2. Modulation of the kinetic properties of enzymes in the aggregated state.

Institut Jacques Monod, CNRS-Université Paris VII, France.
European Journal of Biochemistry (Impact Factor: 3.58). 12/1993; 217(3):1075-82. DOI: 10.1111/j.1432-1033.1993.tb18339.x
Source: PubMed

ABSTRACT The carboxylase activity of free ribulose 1,5-bisphosphate carboxylase-oxygenase has been compared to that of the five-enzyme complex present in chloroplasts. Kinetic results have shown that the V/active site is lower for the free enzyme than for the complex. Conversely the Km is smaller for the complex than for the free enzyme. This implies that the catalytic activity of the enzyme is enhanced when it is embedded in the complex. Under reducing conditions and in the presence of reduced thioredoxin, inactive oxidized phosphoribulokinase, free in solution or inserted in the multi-enzyme complex, becomes active. The kinetics of this activation process has been studied and shown to be exponential. The time constant of this exponential decreases, for the free enzyme, as thioredoxin concentration is increased. Alternatively, for the enzyme embedded in the complex, this time constant increases with thioredoxin concentration almost in a linear fashion. This implies that the complex is much more rapidly activated by reduced thioredoxin than is the free phosphoribulokinase. The variation of the amplitude of this activation process as a function of thioredoxin concentration is a hyperbola. The concentration of thioredoxin which results in half the asymptotic value of this hyperbola is smaller for the complex than for the free enzyme. A kinetic model has been proposed and the dynamic equations resulting from this model have been derived. They fit the experimental results exactly. From the variation of the amplitude of the activation process one may derive the binding constants of thioredoxin on either the oxidized enzyme or on a partly dithiothreitol-reduced enzyme (both of them free or inserted in the complex). In either case, the affinity of reduced thioredoxin is larger for the complex than for the free enzyme. The individual values of some of the rate constants have also been estimated from the variation of the time constants as a function of thioredoxin concentration. Taken together, these results show that at least two enzymes, ribulose 1,5-bisphosphate carboxylase-oxygenase and phosphoribulokinase, have quite different kinetic properties depending on whether they are in free solution or embedded in the multi-enzyme complex.

0 Bookmarks
 · 
54 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Recovery of photosynthesis in rehydrating desiccated leaves of the poikilochlorophyllous desiccation-tolerant plant Xerophyta scabrida was investigated. Detached leaves were remoistened under 12 h light/dark cycles for 96 h. Water, chlorophyll (Chl), and protein contents, Chl fluorescence, photosynthesis-CO(2) concentration response, and the amount and activity of Rubisco were measured at intervals during the rehydration period. Leaf relative water contents reached 87% in 12 h and full turgor in 96 h. Chl synthesis was slower before than after 24 h, and Chla:Chlb ratios changed from 0.13 to 2.6 in 48 h. The maximum quantum efficiency recovered faster during rehydration than the photosystem II operating efficiency and the efficiency factor, which is known to depend mainly on the use of the electron transport chain products. From 24 h to 96 h of rehydration, net carbon fixation was Rubisco limited, rather than electron transport limited. Total Rubisco activity increased during rehydration more than the Rubisco protein content. Desiccated leaves contained, in a close to functional state, more than half the amount of the Rubisco protein present in rehydrated leaves. The results suggest that in X. scabrida leaves Rubisco adopts a special, protective conformation and recovers its activity during rehydration through modifications in redox status.
    Journal of Experimental Botany 10/2010; 62(3):895-905. · 5.24 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Rubisco, the most abundant protein serving as the primary engine generating organic biomass on Earth, is characterized by a low catalytic constant (in higher plants approx. 3s(-1)) and low specificity for CO(2) leading to photorespiration. We analyze here why this enzyme evolved as the main carbon fixation engine. The high concentration of Rubisco exceeding the concentration of its substrate CO(2) by 2-3 orders of magnitude makes application of Michaelis-Menten kinetics invalid and requires alternative kinetic approaches to describe photosynthetic CO(2) assimilation. Efficient operation of Rubisco is supported by a strong flux of CO(2) to the chloroplast stroma provided by fast equilibration of bicarbonate and CO(2) and forwarding the latter to Rubisco reaction centers. The main part of this feedforward mechanism is a thylakoidal carbonic anhydrase associated with photosystem II and pumping CO(2) from the thylakoid lumen in coordination with the rate of electron transport, water splitting and proton gradient across the thylakoid membrane. This steady flux of CO(2) limits photosynthesis at saturating CO(2) concentrations. At low ambient CO(2) and correspondingly limited capacity of the bicarbonate pool in the stroma, its depletion at the sites of Rubisco is relieved by utilizing O(2) instead of CO(2), i.e. by photorespiration, a process which supplies CO(2) back to Rubisco and buffers the redox state and energy level in the chloroplast. Thus, the regulation of Rubisco function aims to keep steady non-equilibrium levels of CO(2), NADPH/NADP and ATP/ADP in the chloroplast stroma and to optimize the condition of homeostatic photosynthetic flux of matter and energy.
    Bio Systems 12/2011; 107(3):158-66. · 1.27 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: An electrophoretically homogeneous preparation of a multienzyme complex with a mol wt of 520 ± 20 kD was isolated from 15- to 25-day-old cotton (Gossypium hirsutum L.) leaves uniform in their age and position on the shoot (3rd to 4th leaves from the top when the plants possessed 5–6 true leaves). When cotton leaves were sampled at the stage of budding and flowering, two complexes with mol wts of 520 ± 20 kD and 480 ± 15 kD were isolated. Comparative enzymatic studies of these complexes revealed developmental changes in activities of ribose phosphate isomerase, phosphoribulokinase, and ribulose-bisphosphate carboxylase. The multienzyme complexes were almost identical in activity of ribose phosphate isomerase. Activities of phosphoribulokinase and ribulose-bisphosphate carboxylase for various multienzyme complexes showed variations between 14 and 17% in the presence of ribose-5-phospate + ATP as a substrate, but the differences were smaller (6–7%) in the presence of specific substrates of these enzymes. The formation of multienzyme Benson-Calvin cycle complexes, featuring different properties at various stages of plant development, is presumably related to the increased demand in assimilates for epigenetic processes during formation of generative organs. Key wordsGossypium hirsutum-multienzyme complexes-Benson-Calvin cycle-development-enzyme activity
    Russian Journal of Plant Physiology 01/2010; 57(2):175-180. · 0.62 Impact Factor