title: Role of phenylalanine-327 in the closure of loop 6 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Cambridge, England.
authors.forename: A G
authors.initials: AG
authors.lastname: Day
authors.forename: P
authors.initials: P
authors.lastname: Chène
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D010649:Phenylalanine; D012273:Ribulose-Bisphosphate Carboxylase