Day A G, Chène P, Fersht A R
Department of Chemistry, University of Cambridge, England.
Biochemistry. 1993 Mar 2;32(8):1940-4. doi: 10.1021/bi00059a009.
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 kcat and the CO2/O2 specificity are unchanged while Km(RuBP) (RuBP = ribulose 1,5-bisphosphate) is drastically increased. The mutant enzyme with the highest value for Km(RuBP),Phe327-->Gly, shows a 165-fold increase (1160 microM compared to 7 microM for the wild-type). The increase in Km(RuBP) suggests an alteration of the ratio kon/koff 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.
