In vitro assay of the chlorophyll biosynthetic enzyme Mg-chelatase: resolution of the activity into soluble and membrane-bound fractions.

Abstract

The fist committed step in chlorophyll synthesis is the Mg-chelatase-catalyzed insertion of magnesium into protoporphyrin IX. Since iron insertion into protoporphyrin leads to heme formation, Mg-chelastase lies at the branch point of heme and chlorophyll synthesis in chloroplasts. Little is known about the enzymology or regulation of Mg-chelatase, as it has been assayed only in intact cucumber chloroplasts. In this report we describe an in vitro assay for Mg-chelatase.Mg-chelatase activity in intact pea chloroplasts was 3-to 4-fold higher than in cucumber chloroplasts. This activity survived chloroplast lysis and could be fractionated by centrifugation into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity, and both were inactivated by boiling indicating that the enzyme is composed of soluble and membrane-bound protein(s). The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. The specific activity of the reconstituted system was typically 1 nmol of Mg-deuteroporphyrin per h per mg of protein, and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity and the enzyme was sensitive to the sulfhydryl reagent N-ethyhnaleimide (I50, 20 ,uM). Broken and reconstituted cucumber chloroplasts were unable to maintain Mg-chelatase activity. However, the cucumber supernatant fraction was active when combined with the pellet fraction of peas; the converse was not

Mechanism and regulation of Mg-chelatase

“…In contrast, Mg-chelatase has resisted attempts at characterization for many years and has proved to be a much more difficult enzyme to analyse. The major impediment was the lack of an in itro assay, which was only achieved 6 years ago [2]. Since that time our knowledge of Mg-chelatase has expanded dramatically to the extent that the enzyme has been cloned and sequenced from several photosynthetic species [3][4][5][6][7][8][9][10].…”

“…The reason for the loss of activity on breakage became apparent when high in itro levels of Mg-chelatase activity were reported for lysed pea (Pisum sati um) chloroplasts [2]. The key to preserving activity was to lyse chloroplasts at a high protein concentration so as not to dilute the chloroplast contents.…”

“…The in itro assay of Walker and Weinstein [2] established that a minimum of two proteins were required for Mg-chelatase activity. Since either the membrane or the soluble fraction could contain multiple Mg-chelatase subunits, the exact number of individual proteins or subunits involved in the reaction could not be established.…”

A Putative Mg Chelatase Subunit from Arabidopsis thaliana cv C24 (Sequence and Transcript Analysis of the Gene, Import of the Protein into Chloroplasts, and in Situ Localization of the Transcript and Protein

“…Walker and Weinstein (1991b) demonstrated that Mg chelatase activity required soluble and membrane fractions of lysed pea chloroplasts. They later reported that Mg chelatase activity was rendered entirely soluble if pea chloroplasts were lysed in buffers containing low concentrations of Mg 2+ (Walker and Weinstein, 1995).…”

“…Accordingly, this enzyme has been the subject of much study in plants (Fuesler et al, 1981;Weinstein 1991a, 1991b;Walker and Weinstein, 1994), although purification of the enzyme has proved difficult. At present, it has been possible to establish a continuous assay for the enzyme and to show that two components probably participate in the chelation reaction (Walker and Weinstein, 1991b;Walker and Weinstein, 1995). It is also known that there is an ATP requirement for the activation of these components (Walker and Weinstein, 1994).…”