Plant Physiology 95:1189-1196 (1991)
© 1991 American Society of Plant Biologists
Metabolism and Enzymology
Further Characterization of the Magnesium Chelatase in Isolated Developing Cucumber Chloroplasts 1
Substrate Specificity, Regulation, Intactness, and ATP Requirements
Caroline J. Walker and
Jon D. Weinstein
Department of Biological Sciences, Clemson University, Clemson, South Carolina 29634-1903
Mg-chelatase catalyzes the first step unique to the chlorophyll branch of tetrapyrrole biosynthesis, namely the insertion of Mg into protoporphyrin IX (Proto). Mg-chelatase was assayed in intact chloroplasts from semi-green cucumber (Cucumis sativus, cv Sumter) cotyledons. In the presence of Proto and MgATP, enzyme activity was linear for 50 minutes. Plastid intactness was directly related to (and necessary for) Mg-chelatase activity. Uncouplers and ionophores did not inhibit Mg-Chelatase in the presence of ATP. The nonhydrolyzable ATP analogs,  , -methylene ATP and adenylylimidodiphosphate, could not sustain Mg-chelatase activity alone and were inhibitory in the presence of ATP (I50 10 and 3 millimolar, respectively). Mg-chelatase was also inhibited by N-ethylmaleimide (I50, 50 micromolar) and the metal ion chelators 2,2'-dipyridyl and 1, 10 phenanthroline (but not to the same degree by their nonchelating analogs). In addition to Proto, the following porphyrins acted as Mg-chelatase substrates, giving comparable specific activities: deuteroporphyrin, mesoporphyrin, 2-ethyl, 4-vinyl Proto and 2-vinyl, 4-ethyl Proto. Mg-chelatase activity and freely exchangeable heme levels increased steadily with greening, reaching a maximum and leveling off after 15 hours in the light. Exogenous protochlorophyllide, chlorophyllide, heme, and Mg-Proto had no measurable effect on Mg-chelatase activity. The potent ferrochelatase inhibitors, N-methylmesoporphyrin and N-methylprotoporphyrin, inhibited Mg-chelatase at micromolar concentrations.
1 This research was supported by Department of Energy grant, DE-FG09-89ER 13989.
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