Citrate lyase deacetylase or acetyl-5'-(acyl-carrier protein) enzyme thioester hydrolase (acetate) (EC 3.1.2. -), was purified 3100-fold wilh a yield of 3.8 0;; from cell extracts of Rlzoc~~pseu~~jn~oizus gelurinosu. The final enzyme preparation gave a single protein band upon polyacrylamide-gel electrophoresis in the absence or in the presence of sodium dodecylsulfate. The molecular weight of the native enzyme was estimated by gel filtration to be 14300 1000. Sodium dodecylsulfate/ polyacrylamide gel electrophoresis yielded a molecular weight of 7300 & 600 indicating that the enzyme consisted of two subunits.Citrate lyase deacetylase acted as an S-acetyl cnzyme thioesterhydrolase because it catalyzed the conversion of citrate lyase (S-acetyl form) into citrate lyase (sulfhydryl form) and acetate.Citrate lyase deacetylase was strongly inhibited by L-glutamate. The half-maximal inhibitor concentration was 7.5 x M, and a Ki-value of 1.2 x lop4 M was determined. The mode of inhibition appeared to be of the linear mixed type. L-Glutamate was bound by citrate lyase deacetylase but not by citrate lyase.The pool concentrations of r2-glutamate in R. gdrrinosu were 10 niM when citrate was present in substrate amounts and 2.7 mM after total consumption of citrate. Simulation of conditions in vivo using homogeneous enzyme preparations of citrate lyase and citrate lyase deacetylase, and glutamate concentrations of 10 mM and 2.7 mM respectively, revealed that the rate of citrate lyase inactivation increased from 8 ?fA within 15 min during growth on citrate to 50 06 within 15 min in the absence of citrate.The activity of citrate lyase in Rho~op~rurlo~zonus gel(i/inosci is regulated by covalent modification. Two enzymes are involved in this regulatory process. They catalyze the acetylation and deacetylation of citrate lyase [1,2] . In E. wrogenes the citrate lyase is also inactivated by deacetylation under certain conditions but this process is energy-dependent and is not catalyzed by a soluble deacetylase [6]. In this publication the purification of citrate lyase deacetylase, some of its kinetic properties and its regulation by 1.-glutamate are reported.
An isolate of Rhodopseudomonas sphaeroides was capable of growing phototrophically and chemotrophically (μ = 0.15 h −1 for either condition) with d -(–)-tartrate as the carbon source. A d -(–)-tartrate dehydratase, ( d -(–)-tartrate hydrolyase, EC 4.1.2.70) was induced in the presence of d -(–)-tartrate. The enzyme was purified 30-fold from cell extracts of R. sphaeroides to a specific activity of 7.5 U/mg of protein and was subsequently crystallized in the presence of 1 M KCl. The enzyme was homogeneous upon analytical electrophoresis in 5% polyacrylamide gels and by criteria of ultracentrifugation. The native enzyme had a molecular weight of 158,000 ± 1,000 as determined by gel filtration and ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single polypeptide chain with an estimated molecular weight of 39,500 ± 500, indicating that d -(–)-tartrate dehydratase was a tetramer. The isoelectric point of the native enzyme was at pH 5.5. The enzyme catalyzed irreversibly the conversion of d -(–)-tartrate to oxaloacetate and water, and the turnover number was calculated to be 1,185. The reaction followed Michaelis-Menten kinetics, and a K m value of 1.8 × 10 −4 M was determined. d -(–)-Tartrate dehydratase required Mg 2+ for activity. The pH optimum was within a range from 6.2 to 7.2, and the activation energy of the reaction (Δ H 0 ) was 63.2 kJ/mol. The enzyme was specific for d -(–)-tartrate; it did not react with l -(+)-tartrate, meso -tartrate, and other hydroxycarboxylic acids. d -(–)-Tartrate dehydratase was strongly inhibited by meso -tartrate (50% at 0.6 mM). l -(+)-Tartrate and a variety of hydroxycarboxylic acids caused 50% inhibition at concentrations of >30 mM.
Of 10 strains of the purple non-sulfur bacterium Rhodopseudomonas sphaeroides , 8 acquired the ability to grow on d -(—)-tartrate; however, growth occurred only after extended lag phases ranging from 2 to 14 days. These lag phases occurred because only a small number of inoculum cells were able to grow by forming the enzyme d -(—)-tartrate dehydratase [ d -(—)-tartrate hydro-lyase; EC number not yet available]. Once cells had grown on d -(—)-tartrate, d -(—)-tartrate dehydratase was formed constitutively. Therefore, mass cultivation of R. sphaeroides for production of large quantities of enzyme was possible on substrates much cheaper than d -(—)-tartrate. When 0.38 mol of dl -malate was used as a substrate in a chemotrophic fed-batch culture, a final biomass of 15 g (dry weight) liter −1 and 1,500 U of d -(—)-tartrate dehydratase liter of culture −1 were formed. The enzyme can be used for selective cleavage of racemic tartaric acid and for quantitative determination of d -(—)-tartrate.
D-(-)-Tartrate dehydratases [D-(-)-tartrate hydro-lyase, EC 4.2.1...] were isolated from two Pseudomonas strains. The molecular weights of the native enzymes were determined to be 72,000 and 78,000, respectively, and each enzyme was composed of two subunits of identical size. The dehydratases had no requirements for thiol compounds, were insensitive to oxygen, and required Fe2+ (0.1 mM) or Co2+ (0.5 mM) ions for optimal activity.
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