PLANT PHYSIOLOGY , Vol 103, Issue 2 501-508, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Covalent Modification of a Highly Reactive and Essential Lysine Residue of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase
M. E. Salvucci
United States Department of Agriculture-Agricultural Research Service, and Agronomy Department, University of Kentucky, Lexington, Kentucky 40546-0076
Chemical modification of ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco) activase with water-soluble N-hydroxy-succinimide esters was used
to identify a reactive lysyl residue that is essential for activity.
Incubation of Rubisco activase with
sulfo-succinimidyl-7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS) or
sulfosuccinimidyl-acetate (sulfo-NHS-acetate) caused progressive
inactivation of ATPase activity and concomitant loss of the ability to
activate Rubisco. AMCA-sulfo-NHS was the more potent inactivator of Rubisco
activase, exhibiting a second-order rate constant for inactivation of 239
M-1 S-1 compared to 21 M-1 S-1 for sulfo-NHS-acetate. Inactivation of
enzyme activity by AMCA-sulfo-NHS correlated with the incorporation of 1.9
mol of AMCA per mol of 42-kD Rubisco activase monomer. ADP, a competitive
inhibitor of Rubisco activase, afforded considerable protection against
inactivation of Rubisco activase and decreased the amount of AMCA
incorporated into the Rubisco activase monomer. Sequence analysis of the
major labeled peptide from AMCA-sulfo-NHS-modified enzyme showed that the
primary site of modification was lysine-247 (K247) in the tetrapeptide
methionine-glutamic acid-lysine-phenylalanine. Upon complete inactivation
of ATPase activity, modification of K247 accounted for 1 mol of AMCA
incorporated per mol of Rubisco activase monomer. Photoaffinity labeling of
AMCA-sulfo-NHS- and sulfo-NHS-acetate-modified Rubisco activase with ATP
analogs derivatized on either the adenine base or on the [gamma]-phosphate
showed that K247 is not essential for the binding of adenine nucleotides
per se. Instead, the data indicated that the essentiality of K247 is
probably due to an involvement of this highly reactive, species-invariant
residue in an obligatory interaction that occurs between the protein and
the nucleotide phosphate during catalysis.
