The Acyl‐Carrier Protein of Citrate Lyase

Abstract

When [14C]acetyl citrate lyase was dissociated in sodium dodecylsulfate and the products were separated by gel electrophoresis, all of the radioactive label appeared in a small protein. This had a molecular weight of about 10000 and contained phosphopantetheine. It was concluded, therefore, that this protein component represents the acyl carrier protein of citrate lyase. [14C]Acetylated acyl carrier protein was purified by conventional methods to homogeneity as judged from the results of gel electrophoretic analysis. The well‐known dissociation of citrate lyase into subunits and their recombination were studied in detail for optimal conditions. It was found that inactivated, deacetylated citrate lyase could also undergo the dissociation‐association process. Recombination of this inactivated and dissociated lyase species in presence of the purified [14C]acetyl acyl carrier protein produced enzymatically active citrate lyase. The biological activity of the lyase acyl carrier protein thus demonstrated was confirmed by correlating enzymic activity and radioactive label of the reconstituted complex. An acyl carrier protein present in crude extracts of Klebsiella aerogenes, but not the deacetylated, purified lyase acyl carrier protein, was active in the malonyl‐CoA‐CO2 exchange reaction catalyzed by an Escherichia coli fatty acid synthetase preparation lacking acyl carrier protein. It was concluded, therefore, that two different acyl carrier proteins are present in K. aerogenes, one for fatty acid synthetase, the other for citrate lyase. Based on experimental results a model is proposed for the construction of citrate lyase. It was assumed that four moles of acyl carrier protein are present in one mole of the native complex which is additionally composed of 16 protein subunits of identical or nearly identical size. The basic functional unit appears to be a pentamer containing one acyl carrier protein and four of these protein subunits. Hence the native complex would consist of a tetramer of the functional basic pentamers. Some of the properties of the lyase acyl carrier protein are described. A remarkable feature is the presence of two sulfhydryl groups, one contributed by a cysteamine, the other by a cysteine residue. Both groups were shown to be acetylated in the isolated lyase acyl carrier protein by labelling experiments performed with iodo[14C]acetate. Whereas these results amply demonstrate the thioester linkage of the acetyl group to acyl carrier protein, the question whether both of the thioesters are enzymically active has not yet been clarified.