A procedure has been worked out which allowed the purification and crystallization of a citrate lyase/citrate lyase ligase complex from Rhodopseudomonas gelatinosa. The complex was subsequently separated to yield two homogeneous enzymes.Citrate lyase ligase was purified 365-fold with a yield of 3.23%. The molecular weight of the enzyme was estimated to be 39500, the enzyme consisted of one polypeptide chain. The reaction rates for ATP, acetate and citrate lyase (sulfhydryl form) followed Michaelis-Menten kinetics ( K , values: 0.14 mM, 5 mM and 37 nM respectively).Citrate lyase ligase exhibited a high substrate specificity and could not react with citrate lyases from nonphototrophic microorganisms. In contrast to the ligase from Streptococcus diucetiluctis, the enzyme from R . gelatinosa was extremely labile ; however, it could be stabilized by nucleotides, the most potent stabilizing one being ADP.The utilization of citrate by Rhodopseudomonas gelatinosa is initiated by its breakdown to oxaloacetate and acetate as catalyzed by the enzyme citrate lyase [l, 21. Previous studies on this enzyme have shown that it is regulated by acetylation and deacetylation [3]. This modification proceeds at the sulfhydryl group of a coenzyme A derivative bound to the smallest subunit type present in citrate lyase [4,5]. The regulatory enzyme which catalyzes the deacetylation of citrate lyase has been isolated from R. gelatinosa and characterized [6]. Little is known on the acetylating enzyme, citrate lyase ligase of R . gelatinosa. Its presence could be demonstrated in extracts of cells actively degrading citrate. Following the acetylation of all the citrate lyase present in the cells it was inactivated rather rapidly [7].For an understanding of the citrate lyase regulatory system it seemed necessary to find conditions for the purification of citrate lyase ligase and to study its properties. Purification can now be achieved by taking advantage of the fact that citrate lyase and citrate lyase ligase form a complex under certain conditions.
MATERIALS AND METHODS
Organism and Method of CultivationRhodopseudomonas gelatinosa strain DSM 149 was grown with citrate anaerobically in the light as described [7]. After 24 h of growth, cells of four 20-1 cultures were harvested and resuspended in 20-1 medium without citrate. After incubation of this suspension for 1 h with magnetic stirring under anaerobic conditions at 5000 lx, sodium citrate was added to give a final concentration of 10 mM and incubation was continued until the citrate concentration was approximately 2 mM. Cells were harvested by centrifugation at 20000 x g for 30 min at 4 "C and kept frozen at -20 "C until needed. The yield of cells from 80-1 culture suspension was 120-150 g (wet weight).
Enzyme AssayEnzyme assays were carried out in cuvettes of 1 cm path length at 30 "C using a Zeiss spectrophotometer. Citrate lyase activity was determined as in [8], and citrate was determined as in [9]. The incubation mixture for the determination of citrate lyase ligase activity contai...