Citrate Lyase Deacetylase of <i>Rhodopseudomonas gelatinosa</i>. Isolation of the Enzyme and Studies on the Inhibition by <scp>l</scp>‐Glutamate

European Journal of Biochemistry

Gottschalk

1982

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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...

Section: Discussionmentioning

confidence: 99%

“…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 .…”

mentioning

confidence: 99%

Copurification of Citrate Lyase and Citrate Lyase Ligase from Rhodopseudomonas gelatinosa and Subsequent Separation of the Two Enzymes

European Journal of Biochemistry

Gottschalk

1982

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“…On the other hand, in Rc. gelatinosus, citrate lyase activity is indirectly influenced by L-glutamate which inhibits citrate lyase inactivating enzyme (citrate lyase deacetylase) [20]. It is the intention of this review to discuss the physiological and biochemical interactions of citrate lyase regulation in different groups of microorganisms.…”

Section: {4)mentioning

confidence: 99%

Citrate metabolism in anaerobic bacteria

FEMS Microbiology Letters

Giffhorn

1987

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The regulation of anaerobic citrate metabolism is very diverse among different groups of bacteria. In organisms like Streptococcus lactis and Clostridium sporosphaeroides which lack citrate synthase, the activity of its antagonistic enzyme, citrate lyase, need not be regulated. Many anaerobes like Rhodocyclus gelatinosus and Clostridium sphenoides are able to synthesize their own l‐glutamate and contain citrate synthase. In these bacteria the activity of citrate metabolizing enzymes which are involved in a cascade system are under strict control. In Rc. gelatinosus activation/inactivation of citrate lyase is controlled by acetylation/deacetylation which is catalyzed by its corresponding regulatory enzymes, citrate lyase ligase and citrate lyase deacetylase. In C. sphenoides inactivation of citrate lyase is accomplished by deacetylation as well as by changing in the enzyme conformation. Activation of citrate lyase is catalyzed by citrate lyase ligase whose activity in addition is modulated by phosphorylation/dephosphorylation. Further, electron transport process also seems to play a role in the inactivation of citrate metabolizing enzymes in enteric bacteria.

“…In R. gelatinosus a futile cycle is avoided by the deacetylation (inactivation) of citrate lyase after the exhaustion of citrate in the medium. The enzyme catalyzing this reaction is citrate lyase deacetylase, whose activity is strongly inhibited by L-glutamate [13].After citrate exhaustion another futile cycle has to be avoided, the one between citrate lyase deacetylase and citrate lyase ligase. The latter enzyme has been shown to be rapidly inactivated in R .…”

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confidence: 99%

mentioning

confidence: 99%

Covalent modification of citrate lyase ligase from Clostridium sphenoides by phosphorylation/dephosphorylation

European Journal of Biochemistry

Herzberg

Gottschalk

1985

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Citrate lyase ligase was shown to be present in Clostridium sphenoides actively degrading citrate. In contrast to citrate lyase ligase from C. sporosphaeroides and Streptococcus lactis, the enzyme from C. sphenoides was under stringent regulatory control. The alteration of the kinetic properties of the enzyme after depletion of citrate suggested the presence of two different enzyme species in different phases of growth: active and partially active citrate lyase ligase. These enzymes were purified from in vivo 32P-labeled C. sphenoides cells, which were grown on low-phosphate medium containing 40 mM citrate and 1 mCi [32P]orthophosphate. During enzyme purification only the active form of citrate lyase ligase was shown to be radioactively labeled. Growth experiments with 14C-labeled precursors of purines and pyrimidines and subsequent purification of active citrate lyase ligase indicated that the 32P labeling of the enzyme was not due to the incorporation of a nucleotide. Inactivation of the ligase after its treatment with acid phosphatase also suggested that the active form of the enzyme is phosphorylated. Citrate lyase ligase, therefore, is the first known enzyme in an anaerobic bacterium whose activity is modulated by phosphorylation/dephosphorylation. both organisms that the intracellular pool size of L-glutamate is dependent on the level of its precursor, citrate, in the medium [12, 131. It is, therefore, reasonable that L-glutamate plays a key role in the regulation of citrate metabolism in these organisms. A high concentration of L-glutamate signals that citrate is available and that it can be cleaved via the fermentation pathway, while at low concentrations citrate is utilized for the biosynthesis of L-glutamate. In R. gelatinosus a futile cycle is avoided by the deacetylation (inactivation) of citrate lyase after the exhaustion of citrate in the medium. The enzyme catalyzing this reaction is citrate lyase deacetylase, whose activity is strongly inhibited by L-glutamate [13].After citrate exhaustion another futile cycle has to be avoided, the one between citrate lyase deacetylase and citrate lyase ligase. The latter enzyme has been shown to be rapidly inactivated in R . gelatinosus [9]. A similar inactivation has been observed in C. sphenoides and it has now been studied in detail in this organism. In this communication we present conclusive evidence that a covalent modification of citrate lyase ligase occurs and that this modification consists of a phosphorylation/dephosphorylation of this enzyme. MATERIALS AND METHODS Organism and growth conditionsClostridium sphenoides strain C 2 (DSM 614) was used for this investigation. The growth medium contained 40 mM trisodium citrate and was prepared as described in [12].Before the total consumption of citrate (17 h) growth was stopped and the harvested cells were stored at -20°C (yield 2.5-3.5 g wet weight/l). In this phase of growth, citrate lyase ligase was present in its active form. For the purification of partially active citrate lyase ligase cells were harv...