D-(--)-tartrate dehydratase of Rhodopseudomonas sphaeroides: purification, characterization, and application to enzymatic determination of D-(--)-tartrate.

Journal of Bacteriology
H Rode, F Giffhorn

Abstract

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-(-)-Tartrat...Continue Reading

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Citations

Nov 7, 1996·Chemical Reviews·Dennis H. Flint, Ronda M. Allen

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