Purification and properties of citraconase

Abstract

The enzyme citraconase (citramalate hydro-lyase, citraconate hydratase, EC 4.2.1) which catalyses the hydration of citraconate to levorotatory citramalate has been purified about 25-fold from an initial specific activity of 0.2 to a final specific activity of 5.0. The enzyme requires activation by a sulfhydryl compound (such as reduced glutathione, cysteine, thioglycollate, or mercaptoethanol) and ferrous ions for maximal activity; ascorbate has no activating effect. The activation is carried out by preincubation of the enzyme and cofactors anaerobically at room temperature. Only ferrous ions, among the metallic ions tested, activate citraconase in conjunction with glutathione. Cupric and mercuric ions inhibit the enzyme activity under similar conditions to the extent of about 80%. The optimum pH for citraconase activity is 7.5 and the activity falls off markedly on both sides of the optimum. The partially purified citraconase preparation does not act on other substrates such as mesaconate, maleate, fumarate, cis- and trans-aconitate. It does not contain any other related enzymic activity except very low isocitrate dehydrogenase activity. The K_m for citraconate at pH 7.5 and 30° is 1.1 mm. Metal-complexing agents (1 mm) such as pyrophosphate, ethylenediaminetetraacetate, o-phenanthroline, 8-hydroxyquinoline and α,α'-bipyridyl have a pronounced (99%) inhibitory effect on the enzyme (in the absence of reduced glutathione). In the presence of 0.5 mm reduced glutathione the inhibition by these agents is of the order of 74% (indicating that glutathione perhaps supplies Fe⁺⁺). Sulfhydryl reagents such as iodoacetate and p-hydroxymercuribenzoate markedly inhibit citraconase activity (98%).