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

Ribo Guo², Meizhong Luo, and Jon D. Weinstein^*

Department of Biological Sciences, Clemson University, Clemson, South Carolina 29634-1903

Mg-chelatase catalyzes the ATP-dependent insertion of Mg²⁺ 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 min¹ mg¹) 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 Mg²⁺ were sigmoidal, with apparent K_m values for Mg²⁺ 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.

Plant Physiol. (1998) 116: 605-615
Copyright Clearance Center:   0032-0889/98/116/0605/11
© 1998 American Society of Plant Physiologists

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