COVALENT MODIFICATION OF A HIGHLY REACTIVE AND ESSENTIAL LYSINE RESIDUE OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE - OXYGENASE ACTIVASE

Chemical modification of ribulose-1,5-bisphosphate carboxylase/oxygena se (Rubisco) activase with water-soluble N-hydroxysuccinimide esters w as used to identify a reactive lysyl residue that is essential for act ivity. Incubation of Rubisco activase with lfosuccinimidyl-7-amino-4-m ethylcoumarin-3-acetate AMCA-sulfo-NHS) or sulfosuccinimidyl-acetate(s ulfo-NHS-acetate) caused progressive inactivation of ATPase activity a nd concomitant loss of the ability to activate Rubisco. AMCA-sulfo-NHS was the more potent inactivator of Rubisco activase, exhibiting a sec ond-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 AMC A-sulfo-NHS correlated with the incorporation of 1.9 mol of AMCA per m ol of 42-kD Rubisco activase monomer. ADP, a competitive inhibitor of Rubisco activase, afforded considerable protection against inactivatio n of Rubisco activase and decreased the amount of AMCA incorporated in to the Rubisco activase monomer. Sequence analysis of the major labele d peptide from AMCA-sulfo-NHS-modified enzyme showed that the primary site of modification was lysine-247 (K247) in the tetrapeptide methion ine-glutamic acidlysine-phenylalanine. Upon complete inactivation of A TPase activity, modification of K247 accounted for 1 mol of AMCA incor porated 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-pho sphate showed that K247 is not essential for the binding of adenine nu cleotides per se. Instead, the data indicated that the essentiality of K247 is probably due to an involvement of this highly reactive, speci es-invariant residue in an obligatory interaction that occurs between the protein and the nucleotide phosphate during catalysis.