PLANT PHYSIOLOGY , Vol 109, Issue 3 927-935, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Isolation and Characterization of Biotin Carboxylase from Pea Chloroplasts
C. Alban, J. Jullien, D. Job and R. Douce
Unite Mixte Centre National de la Recherche Scientifique/Rhone-Poulenc, U.M. 41, Rhone-Poulenc Agrochimie, Lyon, France
Pea (Pisum sativum L.) leaf acetyl-coenzyme A carboxylase (ACCase) exists
as two structurally different forms: a major, chloroplastic, dissociable
form and a minor, multifunctional enzyme form located in the leaf
epidermis. The dissociable form is able to carboxylate free D-biotin as an
alternate substrate in place of the natural substrate, biotin carboxyl
carrier protein. Here we report the purification of the biotin carboxylase
component of the chloroplastic pea leaf ACCase. The purified enzyme, free
from carboxyltransferase activity, is composed of two firmly bound
polypeptides, one of which (38 kD) is biotinylated. In contrast to
bacterial biotin carboxylase, which retains full activity upon removal of
the biotin carboxyl carrier component, attempts to dissociate the two
subunits of the plant complex led to a complete loss of biotin carboxylase
activity. Steady-state kinetic studies of the biotin carboxylase reaction
reveal that addition of all substrates on the enzyme is sequential and that
no product release is possible until all three substrates (MgATP, D-biotin,
bicarbonate) are bound to the enzyme and all chemical processes at the
active site are completed. In agreement with this mechanism,
bicarbonate-dependent ATP hydrolysis by the enzyme is found to be strictly
dependent on the presence of exogenous D-biotin in the reaction medium.
