Effect of mutation of lysine-128 of the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans

The contribution of lysine-128 within the active site of Anacystis nidulans D-ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) w as investigated by the characterization of mutants in which lysine-128 was replaced with arginine, glycine, glutamine, histidine or aspartic acid. Mut ated genes encoding the Rubisco large subunit were expressed in Escherichia coli and the resultant polypeptides assembled into active complexes. All o f the mutant enzymes had a lower affinity for ribulose 1,5-bisphosphate (Ru BP) and lower rates of carboxylation. Substitution of lysine-128 with gluta mine, histidine or aspartic acid decreased the specificity factor and led t o the production of an additional monophosphate reaction product. We show t hat this product results from the loss of the phosphate from C-l of RuBP, m ost probably by beta-elimination from the 2,3-enediolate derivative of RuBP , The results confirm that lysine-128 is important in determining the posit ion of the essential epsilon-amino group of lysine-334 within the active si te and in loop dynamics. This further demonstrates that residues remote fro m the active site can be manipulated to modify catalytic function.