Further Characterization of the Magnesium Chelatase in Isolated Developing Cucumber Chloroplasts 1 Substrate Specificity, Regulation, Intactness, and ATP Requirements

• Published in: Plant physiology (Bethesda) Vol. 95; no. 4; pp. 1189 - 1196
• Main Authors: Walker, Caroline J., Weinstein, Jon D.
• Format: Journal Article
• Language: English
• Published: 01.04.1991
• Subjects: Metabolism and Enzymology
• ISSN: 0032-0889; 1532-2548

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, β,γ-methylene ATP and adenylylimidodiphosphate, could not sustain Mg-chelatase activity alone and were inhibitory in the presence of ATP (I 50 10 and 3 millimolar, respectively). Mg-chelatase was also inhibited by N -ethylmaleimide (I 50 , 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.