title: 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.
authors.affiliation: Department of Biological Sciences, Clemson University, SC 29634-1903.
authors.forename: C J
authors.initials: CJ
authors.lastname: Walker
authors.forename: J D
authors.initials: JD
authors.lastname: Weinstein
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D002734:Chlorophyll / Q000096:biosynthesis*; D003080:Cold Temperature; D004789:Enzyme Activation; D007887:Fabaceae; D008190:Lyases / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D010946:Plants, Medicinal
chemical_list: D010940:Plant Proteins; D002734:Chlorophyll; D000255:Adenosine Triphosphate; D008190:Lyases; C086788:magnesium chelatase; D008274:Magnesium