ROLE OF PHENYLALANINE-327 IN THE CLOSURE OF LOOP-6 OF RIBULOSEBISPHOSPHATE CARBOXYLASE OXYGENASE FROM RHODOSPIRILLUM-RUBRUM

Phenylalanine-327 of ribulosebisphosphate carboxylase/oxygenase (rubisco) from Rhodospirillum rubrum was mutated to tryptophan, leucine, valine, alanine, and glycine, and was also deleted. The least active mutant, the deletion mutant, exhibits less than 0.5% of the carboxylase activity of the wild-type enzyme. Steady-state kinetic analysis of F327-->Leu, Val, Ala, Gly mutant enzymes reveals that k(cat) and the CO2/O2 specificity are unchanged while K(m(RuBP)) (RuBP = ribulose 1,5-bisphosphate) is drastically increased. The mutant enzyme with the highest value for K(m(RuBP)), Phe327-->Gly, shows a 165-fold increase (1160 muM compared to 7 muM for the wild-type). The increase in K(m(RuBP)) suggests an alteration of the ratio k(on)/k(off) for RuBP. A longer hydrophobic lateral chain and/or the presence of an aromatic ring in the wild-type enzyme and the Phe327-->Trp mutant enzyme could explain a better packing of loop 6 in the closed conformation and thus a tighter binding of RuBP at the active site.