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We found that the Mg-isocitrate complex is the true substrate for pine isocitrate lyase and that magnesium acts as a non-essential activator. Both the non-activated and the activated enzyme forms are catalytically active. Our model is consistent with the presence of two Mg-binding sites with different affinities: an activator site with high affinity in addition to the catalytic site with lower affinity. This may result in a complex, fine regulation of isocitrate lyase activity by magnesium. The affinity of the free enzyme for isocitrate is very low. Moreover, free isocitrate does not bind to the activated enzyme, nor it can yield a catalytically active form by binding to an enzyme species whose catalytic site has already been bound by magnesium.Isocitrate lyase (ICL) catalyzes the reversible cleavage of isocitrate into succinate and glyoxylate. This catalysis requires magnesium at optimal concentrations between 3 mM and 8 mM; at higher concentrations Mg2+ has an inhibitory effect [l]. Roche et al. [2] postulated that Mg2+ binds to the enzyme's catalytic site to yield the active form, which binds the substrate. Recently Malhotra et al. [3], studying the effect of magnesium on ICL activity, have proposed the existence of two classes of Mg-binding sites with different affinities and with negative cooperativity.These scanty data constitute all we know about the role of magnesium in ICL kinetics. In particular it is not yet known whether the true substrate of ICL is free isocitrate or the Mgisocitrate complex, an issue whose resolution is vital for a better understanding of the catalytic mechanism. It is rather surprising that those working on ICL kinetics have never tackled this problem and that indeed they appear to have neglected the well-documented interactions of magnesium with isocitrate and with certain buffers. Moreover, ICL is often reported to be a metalloprotein but it would be more correct to consider it as a metal-activated enzyme [4], since MgZt can easily enough be dissociated from ICL. This paper deals with the results of a kinetic study of ICL reaction in which a metal modifier (magnesium) and a substrate (isocitrate) may combine with the enzyme and with each other [5]. Our aim was to clarify the role of magnesium and to identify the true substrate in ICES reaction. This is the first study of its kind on ICL. MATERIALS AND METHODS Materialsthreo-D,-Isocitrate (monopotassium salt) and DL-isocitrate (trisodium salt) were obtained from Sigma; MgClz. 6 H 2 0 (99.999%) was supplied by Aldrich-Chemie (FRG). All other chemicals were obtained as reported elsewhere [6]. Enzyme purification ICL was purified as described in [6]. The purified enzyme was desalted through Sephadex G-25 PD-10 disposable columns (Pharmacia) equilibrated and eluted with 50 mM triethanolamine pH 7.5, 0.1 mM MgC12, 0.1 mM dithiothreitol. ICL assayEnzyme activity was determined at 25°C in 25 mM triethanolamine pH 7.5 and in 25 mM Hepes pH 7.0 by Dixon and Kornberg's continuous method [7], slightly modified as previously described [6]. I...