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. 1982 Jun;150(3):1061–1068. doi: 10.1128/jb.150.3.1061-1068.1982

d-(–)-Tartrate Dehydratase of Rhodopseudomonas sphaeroides: Purification, Characterization, and Application to Enzymatic Determination of d-(–)-Tartrate

Hergo Rode 1, Friedrich Giffhorn 1
PMCID: PMC216323  PMID: 6978882

Abstract

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 Km value of 1.8 × 10−4 M was determined. d-(–)-Tartrate dehydratase required Mg2+ for activity. The pH optimum was within a range from 6.2 to 7.2, and the activation energy of the reaction (Δ H0) 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.

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Selected References

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