PLANT PHYSIOLOGY , Vol 114, Issue 1 131-136, Copyright © 1997 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Mutations in the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Increase the Formation of the Misfire Product Xylulose-1,5-Bisphosphate
R. Flachmann, G. Zhu, R. G. Jensen and H. J. Bohnert
Department of Biochemistry, University of Arizona, Tucson, Arizona 85721
The small subunit (S) increases the catalytic efficiency of
ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) by
stabilizing the active sites generated by four large subunit (L) dimers.
This stabilization appears to be due to an influence of S on the reaction
intermediate 2,3-enediol, which is formed after the abstraction of a proton
from the substrate ribulose-1,5-bisphosphate. We tested the functional
significance of residues that are conserved among most species in the
carboxy-terminal part of S and analyzed their influence on the kinetic
parameters of Synecho-coccus holoenzymes. The replacements in S (F92S,
Q99G, and P108L) resulted in catalytic activities ranging from 95 to 43% of
wild type. The specificity factors for the three mutant enzymes were little
affected (90-96% of wild type), but Km(CO2) values increased 0.5- to
2-fold. Mutant enzymes with replacements Q99G and P108L showed increased
mis-protonation, relative to carboxylation, of the 2,3-enediol
intermediate, forming 2 to 3 times more xylulose-1,5-bisphosphate per
ribulose-1,5-bisphosphate utilized than wild-type or F92S enzymes. The
results suggest that specific alterations of the L/S interfaces and of the
hydrophobic core of S are transmitted to the active site by long-range
interactions. S interactions with L may restrict the flexibility of
active-site residues in L.
