title: Mechanistic insights provided by deletion of a flexible loop at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase.
abstract: To evaluate the functions of flexible loop 6 at the active site of Rhodospirillum rubrum D-ribulose-1,5-bisphosphate carboxylase/oxygenase, the loop was truncated by cassette mutagenesis, whereby seven residues of the twelve-residue loop were excised and replaced by two glycyl residues. The purified loop-deletion mutant was totally devoid of carboxylase activity, but retained substantial catalytic competency in the enolization of ribulose bisphosphate (the initial step in the overall carboxylase pathway) and in normal processing of the six-carbon carboxylated intermediate (the terminal steps in the overall carboxylase pathway). Hence, catalytic impairment resides predominantly at the stage of carboxylation of the initial enediol(ate), a conclusion compatible with mechanistic deductions derived from crystallographic analyses. A critical role of loop 6 in the stabilization of the transition state for carboxylation is reinforced by the findings that the loop-deletion mutant displays preferentially compromised affinity for an analogue of the carboxylated intermediate relative to ribulose bisphosphate and that the mutant converts the substrate to a dicarbonyl compound as a consequence of beta-elimination of phosphate from the initial enediol(ate).
authors.affiliation: Protein Engineering Program, Oak Ridge National Laboratory, Tennessee, USA.
authors.forename: E M
authors.initials: EM
authors.lastname: Larson
authors.forename: F W
authors.initials: FW
authors.lastname: Larimer
authors.forename: F C
authors.initials: FC
authors.lastname: Hartman
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: D004247:DNA; D012273:Ribulose-Bisphosphate Carboxylase