STRUCTURAL AND FUNCTIONAL-PROPERTIES OF A MULTIENZYME COMPLEX FROM SPINACH-CHLOROPLASTS .2. MODULATION OF THE KINETIC-PROPERTIES OF ENZYMESIN THE AGGREGATED STATE

The carboxylase activity of tree ribulose 1,5-bisphosphate carboxylase -oxygenase has been compared to that of the five-enzyme complex presen t in chloroplasts. Kinetic results have shown that the V/active site i s lower for the free enzyme than for the complex. Conversely the K(m) 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 reduc ed thioredoxin, inactive oxidized phosphofibulokinase, free in solutio n or inserted in the multi-enzyme complex, becomes active. The kinetic s of this activation process has been studied and shown to be exponent ial. The time constant of this exponential decreases, for the free enz yme, as thioredoxin concentration is increased. Alternatively, for the enzyme embedded in the complex, this time constant increases with thi oredoxin concentration almost in a linear fashion. This implies that t he complex is much more rapidly activated by reduced thioredoxin than is the free phosphoribulokinase. The variation of the amplitude of thi s activation process as a function of thioredoxin concentration is a h yperbola. The concentration of thioredoxin which results in half the a symptotic value of this hyperbola is smaller for the complex than for the free enzyme. A kinetic model has been proposed and the dynamic equ ations resulting from this model have been derived. They fit the exper imental results exactly. From the variation of the amplitude of the ac tivation process one may derive the binding constants of thioredoxin o n either the oxidized enzyme or on a partly dithiothreitol-reduced enz yme (both of them free or inserted in the complex). In either case, th e affinity of reduced thioredoxin is larger for the complex than for t he free enzyme. The individual values of some of the rate constants ha ve also been estimated from the variation of the time constants as a f unction of thioredoxin concentration. Taken together, these results sh ow that at least two enzymes, ribulose 1,5-bisphosphate carboxylase-ox ygenase and phosphoribulokinase, have quite different kinetic properti es depending on whether they are in free solution or embedded in the m ulti-enzyme complex.