Magnesium-Chelatase from Developing Pea Leaves1 Characterization of a Soluble Extract from Chloroplasts and Resolution into Three Required Protein Fractions

• Published in: Plant physiology (Bethesda) Vol. 116; no. 2; pp. 605 - 615
• Main Authors: Guo, Ribo, Luo, Meizhong, Weinstein, Jon D.
• Format: Journal Article
• Language: English
• Published: American Society of Plant Physiologists 01.02.1998
• ISSN: 0032-0889; 1532-2548

Mg-chelatase catalyzes the ATP-dependent insertion of Mg 2+ into protoporphyrin-IX to form Mg-protoporphyrin-IX. This is the first step unique to chlorophyll synthesis, and it lies at the branch point for porphyrin utilization; the other branch leads to heme. Using the stromal fraction of pea ( Pisum sativum L. cv Spring) chloroplasts, we have prepared Mg-chelatase in a highly active (1000 pmol 30 min −1 mg −1 ) and stable form. The reaction had a lag in the time course, which was overcome by preincubation with ATP. The concentration curves for ATP and Mg 2+ were sigmoidal, with apparent K m values for Mg 2+ and ATP of 14.3 and 0.35 mm, respectively. The K m for deuteroporphyrin was 8 nm. This K m is 300 times lower than the published porphyrin K m for ferrochelatase. The soluble extract was separated into three fractions by chromatography on blue agarose, followed by size-selective centrifugal ultrafiltration of the column flow-through. All three fractions were required for activity, clearly demonstrating that the plant Mg-chelatase requires at least three protein components. Additionally, only two of the components were required for activation; both were contained in the flow-through from the blue-agarose column.