title: Partial Purification and Characterization of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit epsilonN-Methyltransferase.
abstract: The large subunit (LS) of tobacco (Nicotiana rustica) ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P(2) carboxylase) contains a trimethyllysyl residue at position 14, whereas this position is unmodified in spinach ribulose-P(2) carboxylase. A protein fraction was isolated from tobacco chloroplasts by rate-zonal centrifugation and anion-exchange fast protein liquid chromatography that catalyzed transfer of methyl groups from S-adenosyl-[methyl-(3)H]-l-methionine to spinach ribulose-P(2) carboxylase. (3)H-Methyl groups incorporated into spinach ribulose-P(2) carboxylase were alkaline stable but could be removed by limited tryptic proteolysis. Reverse-phase high-performance liquid chromatography of the tryptic peptides released after proteolysis showed that the penultimate N-terminal peptide from the LS of spinach ribulose-P(2) carboxylase contained the site of methylation, which was identified as lysine-14. Thus, the methyltransferase activity can be attributed to S-adenosylmethionine:ribulose-P(2) carboxylase LS (lysine) ;N-methyltransferase, a previously undescribed chloroplast enzyme. The partially purified enzyme was specific for ribulose-P(2) carboxylase and exhibited apparent K(m) values of 10 micromolar for S-adenosyl-l-methionine and 18 micromolar for ribulose-P(2) carboxylase, a V(max) of 700 picomoles CH(3) groups transferred per minute per milligram protein, and a broad pH optimum from 8.5 to 10.0. S-Adenosylmethionine:ribulose-P(2) carboxylase LS (lysine)(epsilon)N-methyltransferase was capable of incorporating 24 (3)H-methyl groups per spinach ribulose-P(2) carboxylase holoenzyme, forming 1 mole of trimethyllysine per mole of ribulose-P(2) carboxylase LS, but was inactive on ribulose-P(2) carboxylases that contain a trimethyllysyl residue at position 14 in the LS. The enzyme did not distinguish between activated (Mg(2+) and CO(2)) and unactivated forms of ribulose-P(2) carboxylase as substrates. However, complexes of activated ribulose-P(2) carboxylase with the reaction-intermediate analogue 2'-carboxy-d-arabinitol-1,5-bisphosphate, or unactivated spinach ribulose-P(2) carboxylase with ribulose-1,5-bisphosphate, were poor substrates for tobacco LS (epsilon)N-methyltransferase.
authors: Department of Horticulture and Landscape Architecture, Plant Physiology/Biochemistry/Molecular Biology Program, University of Kentucky, Lexington, Kentucky 40546.
authors: R L
authors: RL
authors: Houtz
authors: M
authors: M
authors: Royer
authors: M E
authors: ME
authors: Salvucci