Purification and properties of uroporphyrinogen decarboxylase from Saccharomyces cerevisiae. Yeast uroporphyrinogen decarboxylase.
Uroporphyrinogen decarboxylase (EC 4.1.1.37) was purified about 14000-fold to homogeneity from the yeast Saccharomyces cerevisiae with a 70% overall yield. The purification included affinity chromatography on uroporphyrin-I-Affi-Gel 102. The specific activity of the final preparation was 1750 nmol coproporphyrinogen formed.h-1.(mg protein)-1 at pH 7.5 and 37 degrees C using 4 microM uroporphyrinogen I as substrate. The purified enzyme has a minimum molecular mass of 38 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 46 kDa by gel filtration, suggesting that yeast uroporphyrinogen decarboxylase is a monomer. Chromatofocusing gave a pI of 6.0. Enzyme activity was inhibited by metals, such as Cu2+, Zn2+, Fe2+, Fe3+ and by sulfhydryl-specific reagents, but no cofactor requirement could be demonstrated. The optimum pH was pH 5.7 for uroporphyrinogens I and III and heptacarboxylate porphyrinogen I as estimated by coproporphyrinogen formation. The optimum pH for substrate decarboxylation was pH 5.7 for uroporphyrinogen I, but pH 6.8 for the two other substrates. The Km values at pH 5.7 were 10 nM for uroporphyrinogen I, 6 nM for uroporphyrinogen III and 7 nM for heptacarboxylate porphyrinogen I as measured by coproporphyrinogen formation. The pattern of accumulation of intermediate and final decarboxylation products and the rates of the successive decarboxylations were determined for the three substrates at different concentrations at pH 5.7 and pH 6.8. The rate-limiting step at 4 microM substrate concentration was the elimination of the second carboxyl group of uroporphyrinogen III and the fourth carboxyl of uroporphyrinogen I. An antiserum to purified yeast uroporphyrinogen decarboxylase was used to characterize the protein in several mutants.
Felix
F
F
Laboratoire de Biochimie des Porphyrines, Institut Jacques Monod, Université Paris 7, France.
Brouillet
N
N
D002262:Carboxy-Lyases / Q000302:isolation & purification*; D002384:Catalysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003305:Coproporphyrinogens / Q000096:biosynthesis; D003653:Decarboxylation; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000096:biosynthesis; D007120:Immunochemistry; D007525:Isoelectric Focusing; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D013050:Spectrometry, Fluorescence; D014575:Uroporphyrinogen Decarboxylase / Q000276:immunology / Q000302:isolation & purification*; D014578:Uroporphyrins
D003305:Coproporphyrinogens; D007106:Immune Sera; D014578:Uroporphyrins; C034103:uroporphyrin I; D002262:Carboxy-Lyases; D014575:Uroporphyrinogen Decarboxylase