RIBULOSE 1,5-DIPHOSPHATE CARBOXYLASE FROM HYDROGENOMONAS EUTROPHA AND HYDROGENOMONAS FACILIS .I. PURIFICATION METALLIC ION REQUIREMENTS INHIBITION AND KINETIC CONSTANTS

Ribulose 1,5-diphosphate carboxylase has been purified to homogeneity from two chemoautotrophic microorganisms, Hydrogenomonas eutropha and Hydrogenomonas facilis. The carboxylase from H. eutropha was purified 26- fold and catalyzed the carboxylation of 1.9 μmoles of D-ribulose 1,5-diphosphate per min per mg of protein at pH 8.0 and 30°. Magnesium ion was the most effective metallic cofactor. Cobaltous ion could partially substitute (11%) for magnesium ion but manganous ion was inactive. Sulfate and orthophosphate did not inhibit the enzyme. The Km for ribulose 1,5-diphosphate was 1.25 × 10-4 M. The carboxylase from H. facilis was purified 20-fold to a specific activity of 1.36 by one procedure. Magnesium ion was the most effective metallic cofactor (Km = 1.39 × 10-3 M). Manganous (18%) and cobaltous (9%) ions could partially substitute for magnesium ion. Both sulfate (Ki = 5.1 × 10-3 M) and orthophosphate (Ki = 10 × 10-3 M) were competitive inhibitors with respect to ribulose 1,5-diphosphate. Km values for ribulose diphosphate and bicarbonate were 2.35 × 10-4 and 4.16 × 10-3 M, respectively. 3-Phosphoglycerate inhibited competitively with respect to bicarbonate (K1 = 15 × 10-3 M) and noncompetitively with respect to ribulose diphosphate (Ki = 14.7 × 10-3 M). Both enzymes were free of contaminating enzyme activities often associated with the enzyme from plant sources. Homogeneous preparations could be stored for more than 4 weeks at 2° in 0.02 M Tris-sulfate, containing 0.01 M MgCk 0.05 M NaHC03, and 1 mM EDTA with no loss in activity. © 1969, American Chemical Society. All rights reserved.