STRUCTURAL AND FUNCTIONAL-PROPERTIES OF A MULTIENZYME COMPLEX FROM SPINACH-CHLOROPLASTS .2. MODULATION OF THE KINETIC-PROPERTIES OF ENZYMESIN THE AGGREGATED STATE
B. Gontero et al., STRUCTURAL AND FUNCTIONAL-PROPERTIES OF A MULTIENZYME COMPLEX FROM SPINACH-CHLOROPLASTS .2. MODULATION OF THE KINETIC-PROPERTIES OF ENZYMESIN THE AGGREGATED STATE, European journal of biochemistry, 217(3), 1993, pp. 1075-1082
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.