PLANT PHYSIOLOGY , Vol 102, Issue 4 1129-1137, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Amino-Terminal Truncations of the Ribulose-Bisphosphate Carboxylase Small Subunit Influence Catalysis and Subunit Interactions
K. Paul, M. K. Morell and T. J. Andrews
Plant Environmental Biology, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra, Australian Capital Territory 2601, Australia
The first 20 residues at the amino terminus of the small subunit of spinach
ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes
extensive contacts with the large subunit and also with another small
subunit (S. Knight, I. Andersson, and C.-I. Branden [1990] J Mol Biol 215:
113-160). The influence of these contacts on subunit binding and,
indirectly, on catalysis was investigated by constructing truncations from
the amino terminus of the small subunit of the highly homologous enzyme
from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the
first six residues (and thus the region of contact with a neighboring small
subunit) affected neither the affinity with which the small subunits bound
to the large subunits nor the catalytic properties of the assembled
holoenzyme. Extending the truncation to include the first 12 residues
(which encroaches into a highly conserved region that interacts with the
large subunit) also did not weaken intersubunit binding appreciably, but it
reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of
an additional single residue (i.e. removal of a total of 13 residues)
weakened intersubunit binding approximately 80-fold. Paradoxically, this
partially restored catalytic activity to approximately 40% of that of the
wild-type holoenzyme. None of these truncations materially affected the Km
values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of
the irregular arm (thereby deleting the conserved region of contact with
large subunits) totally abolished the small subunit's ability to bind to
large subunits to form a stable holoenzyme. However, this truncated small
subunit was still synthesized by the E. coli cells. These data are
interpreted in terms of the role of the amino-terminal arm of the small
subunit in maintaining the structure of the holoenzyme.
