Mutations in the small subunit of ribulosebisphosphate carboxylase affect subunit binding and catalysis.
Fully functional Synechococcus PCC 6301 ribulose 1,5-bisphosphate carboxylase-oxygenase (kcat = 11.8 s-1) was assembled in vitro following separate expression of the large- and small-subunit genes in different Escherichia coli cultures. The small subunits were expressed predominantly as monomers, in contrast to the large subunits which have been shown to be largely octameric when expressed separately [Andrews, T. J. (1988) J. Biol. Chem. 263, 12213-12219]. This separate expression system was applied to the study of mutations in the amino-terminal arm of the small subunit, which is one of the major sites of contact with the large subunit in the assembled hexadecamer. It enabled the effects of a mutation on the tightness of binding of the small subunit to the large-subunit octamer to be distinguished from the effects of the same mutation on catalysis carried out by the assembled complex when fully saturated with mutant small subunits. This important distinction cannot be made when both subunits are expressed together in the same cell. Substitutions of conserved amino acid residues at positions 14 (Ala, Val, Gly, or Asp instead of Thr) and 17 (Cys instead of Tyr), which make important contacts with conserved large-subunit residues, were introduced by site-directed mutagenesis. All mutant small subunits were able to bind to large subunits and form active enzymes. A potential intersubunit hydrogen bond involving the Thr-14 hydroxyl group is shown to be unimportant. However, the binding of Gly-14, Asp-14, and Cys-17 mutant small subunits was weaker, and the resultant mutant enzymes had reduced catalytic rates compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)
Paul
K
K
Research School of Biological Sciences, Australian National University, Canberra.
Morell
M K
MK
Andrews
T J
TJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D000458:Cyanobacteria / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics*; D013329:Structure-Activity Relationship
D012273:Ribulose-Bisphosphate Carboxylase