Abstract
Abstract Coproporphyrinogenase was isolated from mitochondrial extracts of Saccharomyces cerevisiae following mechanical disruption. The enzyme was purified 150-fold by a procedure involving ion exchange chromatography and gel filtration. The molecular weight of the enzyme was 75,000 ± 7,500. The purified enzyme catalyzed the conversion of coproporphyrinogen III to protoporphyrinogen IX under both aerobic and anaerobic conditions. In the presence of air, the conversion was not dependent upon the presence of an exogenous hydrogen acceptor or other cofactors. Under anaerobic conditions, however, coproporphyrinogenase had an absolute requirement for divalent metal ion, ATP, and l-methionine. NAD+ or NADP+ was required as an electron acceptor, the latter being more effective. A pH of 7.6 was optimal for both the aerobic and anaerobic reactions. Apparent Km values of 3.2 x 10-5 m and 2.6 x 10-5 m were determined for the aerobic and anaerobic reactions, respectively. In the presence of high salt concentrations, chelating agents, sulfhydryl reactive compounds, or GSSG both the aerobic and anaerobic coproporphyrinogenase activities were inhibited. Anaerobic activity was also inhibited in the presence of 5 mm GSH or l-ethionine, but neither of these compounds affected the aerobic enzyme activity. Both the aerobic and anaerobic coproporphyrinogenase activities were insensitive to FAD, FMN, 2,4-dinitrophenol, and cyanide.
Highlights
The purified enzyme catalyzed the conversion of coproporphyrinogen III to protoporphyrinogen IX under both aerobic and anaerobic conditions
We describe the partial purification and some of the characteristSics of an anaerobic coproporphyrinogenase from Saccharomyces cerevisiae, u hich, in certain respects, resembled the anaerobic coproporphyrirlogellase found in extracts of several photosyntShetic prokaryotes [12, 13]
One unit of coproporphyrinogenase activity is defined as the amount which catalyzes the formation of 1 nmole of protoporphyrinogen IX from the coproporphyrinogen III, under standard assay conditions
Summary
Coproporphyrinogenase was isolated from mitochondrial extracts of Saccharomyces cereuisiae following mechanical disruption. The enzyme was purified E&fold by a procedure involving ion exchange chromatography and gel filtration. The purified enzyme catalyzed the conversion of coproporphyrinogen III to protoporphyrinogen IX under both aerobic and anaerobic conditions. Coproporphyrinogenase had an absolute requirement for divalent metal ion, ATP, and L-methionine. A pH of 7.6 was optimal for both the aerobic and anaerobic reactions. M and 2.6 x lOA5 M were determined for the aerobic and anaerobic reactions, respectively. In the presence of high salt concentrations, chelating agents, sulfhydryl reactive compounds, or GSSG both the aerobic and anaerobic coproporphyrinogenase activities were inhibited. L-ethionine, but neither of these compounds affected the aerobic enzyme activity. Both the aerobic and anaerobic coproporphyrinogenase activities were insensitive to FAD, FMN, 2,4=dinitrophenol, and cyanide.
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