ISOCITRATE LYASE FROM PSEUDOMONAS INDIGOFERA .V. SUBUNITS AND TERMINAL RESIDUES AND RELATION TO CATALYTIC ACTIVITY

Isocitrate lyase from Pseudomonas indigofera is composed of four subunits. Treatment with carboxypeptidase A results in the rapid release of histidine that approaches a tentative limiting yield of 4 moles/mole of isocitrate lyase. Hydrazinolysis confirms that histidine is carboxy terminal. Phenylalanine may be penultimate. Histidine removal catalyzed by carboxypeptidase A shows excellent concordance with loss in activity of isocitrate lyase. The sedimentation coefficient of so-altered isocitrate lyase is identical with that of native enzyme. Isocitrate lyase is resistant to proteolysis by carboxypeptidase B and both native and urea-treated enzyme are resistant to leucine aminopeptidase. Studies with 1-dimethylaminonaphthalene-5-sulfonyl chloride indicate that methionine is the amino-terminal residue. Isocitrate lyase is irreversibly inactivated by 4.2 mM 2- bromomalonate, an analog of the competitive inhibitor, malonate. Protection against this inactivation is provided by the substrate, isocitrate, at 2.2mM. The pH dependence of inhibition by 2-bromomalonate suggests involvement of nucleophilic groups of an average pKa of 6.8 on isocitrate lyase. Guanidine hydrochloride at 0.8 m rapidly inhibits isocitrate lyase to the extent of 88 % but this is not accompanied by spectral alteration or dissociation of the enzyme. Removal of guanidine hydrochloride through dialysis largely re-verses the inhibition. Onset of inhibition by 2.4 m urea is slower and the inhibition is less extensive. Inhibition by 3 m urea is not reversed by dialysis. Dissociation of isocitrate lyase by 3-6 m guanidine hydrochloride has been studied with the meniscus-depletion ultracentrifugal technique of Yphantis. The subunits have an average molecular weight (uncorrected for preferential interaction of third component) of 48,200. The molecular weight of native enzyme is about 206,000. The physical and chemical data suggest that isocitrate lyase is comprised of four subunits of similar, if not identical, size. This number of subunits has been supported directly with electron microscopy. © 1968, American Chemical Society. All rights reserved.