Partial purification and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit ^εN-methyltransferase

The large subunit (LS) of tobacco (Nicotiana rustica) ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P₂ carboxylase) contains a trimethyllysyl residue at position 14, whereas this position is unmodified in spinach ribulose-P₂ 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-³H]-L-methionine to spinach ribulose-P₂ carboxylase. ³H-Methyl groups incorporated into spinach ribulose-P₂ 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-P₂ carboxylase contained the site of methylation, which was identified as lysine-14. Thus, the methyltransferase activity can be attributed to S-adenosylmethionine:ribulose-P₂ carboxylase LS (lysine) ^εN-methyltransferase, a previously undescribed chloroplast enzyme. The partially purified enzyme was specific for ribulose-P₂ carboxylase and exhibited apparent K_m values of 10 micromolar for S-adenosyl-L-methionine and 18 micromolar for ribulose-P₂ carboxylase, a V_max of 700 picomoles CH₃ groups transferred per minute per milligram protein, and a broad pH optimum from 8.5 to 10.0. S-Adenosylmethionine:ribulose-P₂ carboxylase LS (lysine)^εN-methyltransferase was capable of incorporating 24 ³H-methyl groups per spinach ribulose-P₂ carboxylase holoenzyme, forming 1 mole of trimethyllysine per mole of ribulose-P₂ carboxylase LS, but was inactive on ribulose-P₂ carboxylases that contain a trimethyllysyl residue at position 14 in the LS. The enzyme did not distinguish between activated (Mg²⁺ and CO₂) and unactivated forms of ribulose-P₂ carboxylase as substrates. However, complexes of activated ribulose-P₂ carboxylase with the reaction-intermediate analogue 2′-carboxy-D-arabinitol1,5-bisphosphate, or unactivated spinach ribulose-P₂ carboxylase with ribulose-1,5-bisphosphate, were poor substrates for tobacco LS ^εN-methyltransferase.