PLANT PHYSIOLOGY , Vol 105, Issue 2 671-680, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Purification and Characterization of Acetyl-Coenzyme A Carboxylase from Diclofop-Resistant and -Susceptible Lolium multiflorum
K. J. Evenson, J. W. Gronwald and D. L. Wyse
Department of Agronomy and Plant Genetics (K.J.E., D.L.W.), University of Minnesota, and Plant Science Research Unit, Agricultural Research Service, United States Department of Agriculture (J.W.G.), St. Paul, Minnesota 55108
Acetyl-coenzyme A carboxylase (ACCase) was purified >100-fold (specific
activity 3.5 units mg-1) from leaf tissue of diclofopresistant and
-susceptible biotypes of Lolium multiflorum. As determined by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, the purified fractions
from both biotypes contained a single 206-kD biotinylated polypeptide. The
molecular mass of the native enzyme from both biotypes was approximately
520 kD. In some cases the native dimer from both biotypes dissociated
during gel filtration to form a subunit of approximately 224 kD. The
inclusion of 5% (w/v) polyethylene glycol 3350 (PEG) in the elution buffer
prevented this dissociation. Steady-state substrate kinetics were analyzed
in both the presence and absence of 5% PEG. For ACCase from both biotypes,
addition of PEG increased the velocity 22% and decreased the apparent Km
values for acetyl-coenzyme A (acetyl-CoA), but increased the Km values for
bicarbonate and ATP. In the presence of PEG, the Km values for bicarbonate
and ATP were approximately 35% higher for the enzyme from the susceptible
biotype compared with the resistant enzyme. In the absence of PEG, no
differences in apparent Km values were observed for the enzymes from the
two biotypes. Inhibition constants (Ki app) were determined for CoA,
malonyl-CoA, and diclofop. CoA was an S-hyperbolic (slope
replots)-I-hyperbolic (intercept replots) noncompetitive inhibitor with
respect to acetyl-CoA, with Ki app values of 711 and 795 [mu]M for enzymes
from the resistant and susceptible biotypes, respectively. Malonyl-CoA
competitively inhibited both enzymes (versus acetyl-CoA) with Ki app values
of 140 and 104 [mu]M for ACCase from resistant and susceptible biotypes,
respectively. Diclofop was a linear noncompetitive inhibitor of ACCase from
the susceptible biotype and a nonlinear, or S-hyperbolic-I-hyperbolic,
noncompetitive inhibitor of ACCase from the resistant biotype. For ACCase
from the susceptible biotype the slope (Kis) and intercept (Kii) inhibition
constants for diclofop versus acetyl-CoA were 0.08 and 0.44 [mu]M,
respectively. ACCase from the resistant biotype had a Ki app value of 6.5
[mu]M. At a subsaturating acetyl-CoA concentration of 50 [mu]M, the Hill
coefficients for diclofop binding were 0.61 and 1.2 for ACCase from the
resistant and susceptible biotypes, respectively. The Hill coefficients for
diclofop binding and the inhibitor replots suggest that the resistant form
of ACCase exhibits negative cooperativity in binding diclofop. However, the
possibility that the nonlinear inhibition of ACCase activity by diclofop in
the enzyme fraction isolated from the resistant biotype is due to the
presence of both resistant and susceptible forms of ACCase cannot be
excluded.
