A comparison of the citrate synthases of Escherichia coli and Acinetobacter anitratum

Abstract

Citrate synthase has been purified to homogeneity from a strain of the Gram-negative aerobic bacterium Acinetobacter anitratum in a form which retains its sensitivity to the allosteric inhibitor NADH. In subunit size, amino acid composition, and antigenic reactivity the enzyme shows a marked structural resemblance to the citrate synthase of the Gram-negative facultative anaerobe Escherichia coli. Whereas the E. coli enzyme is subject to a strong, hyperbolic inhibition by NADH (Hill's number n = 1.0, K1 = 2 μM), the A. anitratum enzyme shows a weak, sigmoid response (n = 1.6, I0.5 = 140 μM) to this nucleotide. With E. coli, NADH inhibition is competitive with acetyl-CoA, and noncompetitive with oxaloacetate; with A. anitratum, NADH is noncompetitive with both substrates. Acinetobacter anitratum citrate synthase shows hyperbolic saturation with acetyl-CoA (n = 1.8). The finding of Weitzman and Jones (Nature (London) 219, 270 (1968)) that NADH inhibition of the enzyme from Acinetobacter spp. is reversible by AMP, while that from E. coli is not, is explained by the much greater affinity of the E. coli enzyme for NADH. Unlike E. coli citrate synthase, the A. anitratum enzyme does not react with the sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of denaturation. With a second sulfhydryl reagent, 4,4′-dithiodipyridine (4,4′-PDS), the A. anitratum enzyme reacts with 1 equiv. of subunit; this modification induces a partial activity loss (attributable to a rise in the Km for acetyl-CoA) and an increase in the sensitivity to NADH. With the E. coli enzyme, 4,4′-PDS causes complete inactivation. Acinetobacter anitratum citrate synthase is much more resistant to urea denaturation than the E. coli enzyme is; the resistance of both enzymes to urea is greatly improved in the presence of 1 M KCl. It is suggested that the amino acid sequences of the subunits of the citrate synthases of these two bacteria are about 90% homologous, and that the 10% differences are in key residues, perhaps largely in the subunit contact regions, which account for the differences in allosteric properties.