The magnesium-insertion step of chlorophyll biosynthesis is a two-stage reaction

Abstract

Mg(2+)-chelatase catalyses the first step unique to chlorophyll synthesis, namely the insertion of Mg2+ into protoporphyrin IX. When pea (Pisum sativum L., cv. Spring) chloroplasts are lysed in a buffer lacking Mg2+ and the thylakoids removed by centrifugation, the remaining mixture of light membranes and soluble proteins (LM/S) has high Mg(2+)-chelatase activity. Several lines of evidence are presented to show that the Mg2+ insertion catalysed by this preparation is a two-step reaction consisting of activation followed by Mg2+ chelation. An activated state of Mg(2+)-chelatase is achieved by preincubating LM/S with ATP. The activated state is observed as the elimination of the approx. 6 min lag in the rate of Mg2+ chelation on addition of the porphyrin substrate. The activity of LM/S assayed at low protein concentrations can be greatly enhanced by preincubating at high protein concentrations (12 mg/ml is optimal). This activation effect requires the presence of both LM and S fractions, as well as ATP. Both steps require ATP, but at different concentrations; the first step is optimal at > 0.5 mM (EC50 = 0.3 mM) and the second step is optimal at 0.3 mM (EC50 < 0.2 mM). ATP in the first step could be replaced by ATP[S]; this analogue could not sustain activity in the second step. This activated state was stable for at least 30 min at room temperature, but chilling of preincubated LM/S on ice for 30 min caused an almost complete loss of the activated state.

Mechanism and regulation of Mg-chelatase

“…While the conclusions of Lee et al [18] may initially seem invalid, more recent data has suggested that Mg-chelatase functions as a multicomponent complex [20,21]. If this proves to be the case, Lee et al's data can be reinterpreted to suggest that the presence of high concentrations of Proto, ATP and Mg# + during gentle lysis may stabilise the entire Mg-chelatase complex.…”

“…Therefore, the further discussion of the mechanism of Mg-chelatase will be described in terms of the ' activation ' and ' chelation ' steps. In pea chloroplast extracts, either adenosine 5h-[γ-thio]-PN4 triphosphate (ATP[S]) or ATP could overcome the lag period, implying that either substrate could be utilized for activation [20]. ATP[S] is typically used as a protein kinase substrate, raising the possibility that protein phosphorylation may play a part in the activation process.…”

“…Could the initial activation, which can be catalysed by ATP [S], involve a protein kinase [20] ? With pure, expressed recombinant proteins this idea is now readily testable by the incubation of I and D subunits with radiolabelled ATP [S].…”

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

“…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). However, the identities of these components and the genes that encode them have not been established.…”

“…1). 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).…”