Plant Physiol, November 2000, Vol. 124, pp. 1131-1138
Multisite Inhibition of Pinus pinea Isocitrate
Lyase by Phosphate1
Francesco
Ranaldi,
Paolo
Vanni, and
Eugenio
Giachetti*
Dipartimento di Scienze Biochimiche, Università di Firenze,
Firenze, Italy
Our results show that the phosphate ion is a nonlinear competitive
inhibitor of Pinus pinea isocitrate lyase. In addition, this compound induces a sigmoidal response of the enzyme, which usually
exhibits standard Michaelis-Menten kinetics. This peculiar behavior of
P. pinea isocitrate lyase could be explained by a dimer
(two-site) model, in which phosphate binds cooperatively, but the
affinity of the vacant site for substrate (the magnesium-isocitrate complex) remains the same. As a result, the interaction of phosphate with free enzyme produces an inhibitor-enzyme-inhibitor species that is
of significant importance in determining reaction rate; a possible
regulatory role of the glyoxylate cycle by inorganic phosphate is
suggested. The mode of phosphate inhibition is consistent with both the
mechanism for magnesium ion activation of P. pinea isocitrate lyase and its site heterogeneity. Our results explain the
cooperative effects observed by some authors in kinetic studies of
isocitrate lyase carried out in phosphate buffers and also account for
the higher Km values determined by
using such assay systems. Phosphate buffer should be avoided in
performing isocitrate lyase kinetics.
1
This work was supported by the Ministero
Università Ricerca Technologica, Italy (ex. 60%).
*
Corresponding author; e-mail eugenio.giachetti{at}unifi.it; fax
0039-055-4222725.
© 2000 American Society of Plant Physiologists
