Cyanate modification of essential lysyl residues in the catalytic subunit of tobacco ribulosebisphosphate carboxylase.
Crystalline ribulose-1,5-bisphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) isolated from tobacco (Nicotiana tabacum L.) leaf homogenates is irreversibly inactivated by incubation with potassium cyanate at pH 7.4. The rate of inactivation is pseudo first-order and linearly dependent on reagent concentration. In the presence of ribulosebisphosphate or high levels of CO2 and Mg2+ the rate constant for inactivation is reduced, suggesting that chemical modification occurs in the active site region of the enzyme. In contrast, neither the effector NADPH nor the activator Mg2+ alone significantly affect the rate of inactivation by cyanate; however, NADPH markedly enhances the protective effect of CO2 and Mg2+. Incubation of the carboxylase with potassium [14C] cyanate in the absence or presence of ribulosebisphosphate revealed that the substrate specifically reduces cyanate incorporation into the large catalytic subunits of the enzyme. Analysis of acid hydrolysates of the radioactive carboxylase indicated that the reagent carbamylates both NH2-terminal groups and lysyl residues in the large and small subunits. Comparison of the substrate-protected enzyme with the inactivated carboxylase revealed that ribulosebisphosphate preferentially reduces lysyl modification within the large subunit. The data here presented indicate that inactivation of ribulosebisphosphate carboxylase by cyanate or its reactive tautomer, isocyanic acid, results from the modification of lysyl residues within the catalytic subunit, presumably at the activator and substrate CO2 binding sites on the enzyme.
D002262:Carboxy-Lyases / Q000378:metabolism; D003485:Cyanates; D007700:Kinetics; D008239:Lysine; D010944:Plants / Q000201:enzymology*; D010947:Plants, Toxic; D011485:Protein Binding; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology
D003485:Cyanates; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine