title: Formation of enzyme-bound carbanion intermediate in the isocitrate lyase-catalyzed reaction: enzymatic reaction of tetranitromethane with substrates and its dependence on effector, pH, and metal ions.
abstract: Isocitrate lyase of germinating castor seed endosperm catalyzes the reactions of succinate and of isocitrate (but not of glyoxylate) with tetranitromethane (TNM), giving rise to the nitroform anion (C-(NO2)3), analogous to the reaction of TNM with carbanions (O.P. Malhotra and U.N. Dwivedi, 1984, Ind. J. Biochem. Biophys. 21, 65-67). The kinetics of this reaction have been investigated under a variety of conditions. At a fixed TNM concentration, the initial rate of reaction exhibits a hyperbolic saturation of the enzyme with isocitrate. The reaction with succinate, however, shows "negative cooperativity" in succinate saturation and the data are consistent with the existence of two sets of succinate binding sites of unequal affinity ("tight" and "loose" sites). Equal reaction rates are observed at enzyme-saturating concentrations of succinate and isocitrate. In every case, the rate of reaction is proportional to the TNM concentration. In the presence of alpha-ketoglutarate, hyperbolic saturation curves are obtained for all the substrates (TNM and succinate or TNM and isocitrate). In the presence of this effector the Km of succinate and TNM are independent of the concentration of the second substrate. On the other hand, sets of parallel straight lines are obtained in the double-reciprocal plots for the enzymatic reaction of TNM with isocitrate in the presence of alpha-ketoglutarate. Studies on the effect of pH on the isocitrate lyase-catalyzed reactions of TNM with succinate, TNM with isocitrate, and succinate with glyoxylate in the absence as well as in the presence of alpha-ketoglutarate show that the proton behaves as an uncompetitive inhibitor in all these reactions, suggesting the presence of a "masked" basic group at the enzyme site, which is protonated in the presence of substrate only. The pKa value of this group lies in the range 6.7-6.9. The enzymatic reactions of TNM with succinate and isocitrate exhibit identical Mg2+ ion dependence. From a comparison of the data on the enzymatic reactions of TNM with the corresponding results on the physiological reaction catalyzed by this enzyme, it has been suggested that an ion pair intermediate (E+ X S-, in which E, S, and S- stand for enzyme, succinate, and succinate carbanion, respectively) lies on the pathway of catalysis by isocitrate lyase.
mesh_terms: D002367:Ricinus communis / Q000201:enzymology; D002384:Catalysis; D006038:Glyoxylates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007522:Isocitrate Lyase / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008697:Methane / Q000031:analogs & derivatives*; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D010947:Plants, Toxic; D011485:Protein Binding; D013379:Substrate Specificity; D013386:Succinates / Q000378:metabolism; D013774:Tetranitromethane / Q000378:metabolism*
chemical_list: D006038:Glyoxylates; D007523:Isocitrates; D007656:Ketoglutaric Acids; D013386:Succinates; C034219:isocitric acid; D007652:Oxo-Acid-Lyases; D007522:Isocitrate Lyase; D008274:Magnesium; C031150:glyoxylic acid; D013774:Tetranitromethane; D008697:Methane