Structural and functional properties of a multi-enzyme complex of 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


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.

Download full-text


Available from: Brigitte Gontero, Nov 26, 2014
  • Source
    • "Within the complex, PRK activation by TRX f1 or m1 was not significantly slower than A 4 -GAPDH activation, indicating that activation by reduction of complexed PRK is fast enough to cope with dissociation of the complex. Surprisingly, however , reductive activation of free oxidized PRK required longer times than activation of PRK in the complex and qualitatively similar results were obtained by Gontero et al. (1993) with proteins purified from spinach, well before the discovery of CP12. Clearly, PRK in the complex is locked into a conformation that, although almost inactive, is more receptive to reductive activation than the free oxidized enzyme. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) can form under oxidizing conditions a supramolecular complex with the regulatory protein CP12. Both GAPDH and PRK activities are inhibited within the complex, but they can be fully restored by reduced thioredoxins (TRXs). We have investigated the interactions of eight different chloroplast thioredoxin isoforms (TRX f1, m1, m2, m3, m4, y1, y2, x) with GAPDH (A(4), B(4), and B(8) isoforms), PRK and CP12 (isoform 2), all from Arabidopsis thaliana. In the complex, both A(4)-GAPDH and PRK were promptly activated by TRX f1, or more slowly by TRXs m1 and m2, but all other TRXs were ineffective. Free PRK was regulated by TRX f1, m1, or m2, while B(4)- and B(8)-GAPDH were absolutely specific for TRX f1. Interestingly, reductive activation of PRK caged in the complex was much faster than reductive activation of free oxidized PRK, and activation of A(4)-GAPDH in the complex was much faster (and less demanding in terms of reducing potential) than activation of free oxidized B(4)- or B(8)-GAPDH. It is proposed that CP12-assembled supramolecular complex may represent a reservoir of inhibited enzymes ready to be released in fully active conformation following reduction and dissociation of the complex by TRXs upon the shift from dark to low light. On the contrary, autonomous redox-modulation of GAPDH (B-containing isoforms) would be more suited to conditions of very active photosynthesis.
    Molecular Plant 03/2009; 2(2):259-69. DOI:10.1093/mp/ssn061 · 6.34 Impact Factor
  • Source
    • "Moreover, instead of a prodrug-activating system it might also be possible to use transgenes encoding secreted cytotoxic proteins or cytokines stimulating a tumor-directed immune response. In addition, the mJun/mFos LZiP domains should also be useful for addressing other problems, as, for example, directing partner proteins to a specific subcellular location (Dang et al. 1991), to increase the activity of multienzyme systems by virtue of a physical link between the enzymes (Davidson et al. 1993; Gontero et al. 1993) or to improve the function of heterologous proteins in a gene therapeutic setting by enabling oligomerization (Walczak et al. 1999). "

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Incluye bibligrafía e índice
Show more