D-(--)-tartrate dehydratase of Rhodopseudomonas sphaeroides: purification, characterization, and application to enzymatic determination of D-(--)-tartrate.
An isolate of Rhodopseudomonas sphaeroides was capable of growing phototrophically and chemotrophically (mu = 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 x 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 (Delta 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.
D002413:Cations, Divalent / Q000494:pharmacology; D003460:Crystallization; D006836:Hydro-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013644:Tartrates / Q000378:metabolism*; D013696:Temperature
D002413:Cations, Divalent; D013644:Tartrates; C034710:D-(-)-tartrate dehydratase; D006836:Hydro-Lyases; C029768:tartaric acid