Purification and Kinetic Studies of Beef Liver Cytoplasmic Aconitase

Abstract

Abstract Steady state kinetic analysis has been used to demonstrate the existence of more than one central complex between aconitase and its substrates, and the interconversion of these complexes as a part of the reaction mechanism. Interconversions of this kind are not ordinarily detected by steady state kinetic studies; however, the branched reaction pathway, by which a given product molecule can arise from either of two substrate molecules, predicts different maximum velocities for formation of the product from the two substrates if different central complexes are involved in the two reactions. If only one complex or identical complexes are involved, these maximum velocities will be equal. Michaelis constants were found to be 200 ± 10 µm for citrate, 7.0 ± 0.5 µm for cis-aconitate, and 34 ± 2 µm for threo-ds-isocitrate. The maximum velocities for conversion of a given substrate to a given product (relative to a value of 1.0 for the maximum rate of conversion of citrate to isocitrate) were 1.04 ± 0.07 for the conversion of citrate to aconitate, 1.71 ± 0.12 for isocitrate to aconitate, 1.66 ± 0.12 for isocitrate to citrate, 3.1 ± 0.3 for aconitate to isocitrate, and 3.14 ± 0.24 for aconitate to citrate. These rates indicate that a unique series of central complexes is involved in the interconversion of any two tricarboxylic acids. The fact that the maximum rates of conversion of a given substrate to each of the other products were equal is at present unexplained, and may either be fortuitous or represent some as yet undiscovered characteristic of the actual mechanism. In order to account for all of the observed data, each substrate must combine with the enzyme to give a different central complex, but there may be more central complexes than three.