Magnesium-Chelatase from Developing Pea Leaves1

Abstract

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.The biosynthesis of heme and chlorophyll are integral parts of chloroplast development and the acquisition of photosynthetic capacity. Both end products are required in specific but vastly different amounts and are synthesized within the chloroplast via a common tetrapyrrole biosynthetic pathway (Beale and Weinstein, 1990;Porra, 1997). The branch point for the formation of chlorophyll and heme is the use of Proto by the two enzymes that catalyze metal insertion. Ferrochelatase catalyzes the insertion of Fe 2ϩ into Proto, whereas Mg-chelatase catalyzes the insertion of Mg 2ϩ into Proto. Because the flux of common precursors to the respective end products is likely to be controlled by modulation of the branch-point enzymes, we were interested in the properties of these enzymes. This report describes the properties of a totally soluble enzyme system from pea (Pisum sativum L. cv Spring) chloroplasts that catalyzes the Mg-chelatase reaction.Previous work has shown that Mg-chelatase activity requires ATP (Pardo et al., 1980; Walker and Weinstein, 1991b). Although the reaction is formally similar to the ferrochelatase reaction, insertion of a divalent cation into Proto, there is no ATP requirement for the Fe-insertion reaction. Our previous work has shown that the Mgchelatase reaction requires at least two different protein components (Walker and Weinstein, 1991b; Walker et al., 1992). We also showed that the reaction proceeds by a two-step mechanism, involving activation followed by Mg 2ϩ insertion; both steps require ATP (Walker and Weinstein, 1994). This hypothesis was based on the following observations. There is a lag phase in the kinetics that can be overcome by preincubation of the crude enzyme fraction with ATP before the porphyrin substrate is added. ATP␥S can substitute for ATP in the preincubatio...

Mechanism and regulation of Mg-chelatase

“…However, the activation could be achieved by incubating only the I and D subunits with ATP, while H was only required for the chelation step. This result has recently been confirmed in fractionated pea chloroplasts, where the fractions of the higher-plant I and D counterparts are required for activation [28].…”

“…The third CobT subunit has no sequence similarity to the D subunit of Mg-chelatase. At the enzyme level, the K m values for the Co-chelatase were 85 nM for hydrogenobyrinic acid a,c diamide, 220 µM for ATP [42] which are comparable with K m values of Mg-chelatase of 360 nM or 13 nM for Proto, and 166 µM or 350 µM for ATP (bacterial and plant respectively) [21,28]. The K m value for the Mg# + of the Mg-chelatase is not directly comparable with the K m value for Co# + , since both enzymes utilize ATP as the Mg[ATP complex.…”

“…This is supported by the recent observation that the artificial in i o elevation of Mg-Proto and Mg-Proto monomethyl ester by feeding ALA to pea seedlings caused a 95 % decrease in the initro-measurable Mg-chelatase activity in isolated pea chloroplasts [58]. However, when these intermediates were directly tested in chloroplasts, and more recently in a solubilized Mgchelatase preparation, no evidence for feedback inhibition by Mg-Proto, Mg-Proto monomethyl ester or Pchlide could be found [16,28]. Since these intermediates do not directly inhibit Mg-chelatase, the only explanation for their apparent inhibition in the ALA-feeding experiments is by reducing the amount of one or more of the Mg-chelatase subunits present.…”

“…Mg-chelatase is readily solubilized from membranes in the absence of Mg [5,19,27]. The solubilized proteins have been separated into three fractions by pseudoaffinity chromatography and ultrafiltration [28], and all three fractions are necessary for Mg-chelatase activity.…”

“…In this step, we suggest that I is a dimer which interacts with a D subunit. Since D appears to behave as an aggregate [19,28], this step is shown as involving the dissociation of a single D subunit from the aggregate : a process which may be precipitated by the presence of I dimers and ATP. This step requires ATP or ATP[S] and could involve protein phosphorylation.…”