CHEMICAL MODIFICATION AND ENZYMIC CHARACTERIZATION OF HIGHER PLANT RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE

The bifunctional enzyme ribulosebisphosphate (RuBP) carboxylase/oxygenase (Rubisco; EC 4.1.1.39) provides a common link between photosynthetic CO(,2) fixation and photorespiration in higher plants. In spite of its abundance and physiological importance, relatively little is known concerning the essential amino acids and localization of large subunit binding domains involved in the catalytic functions and metabolite regulation of this key photosynthetic enzyme. Diethylpyrocarbonate (DEP) inactivation of Rubisco at pH 6.0 exhibited rate saturation, a second order rate constant of 2.5 M(\u27-1)s(\u27-1) and a reaction order of 0.92. Excellent protection against inactivation was afforded by the phosphorylated substrate RuBP and the competitive inhibitors sedoheptulosebisphosphate and inorganic phosphate. The formation of ethoxyformyl histidine residues was accompanied by an increase in absorbance at 239 nm ((DELTA)A(,239)). Verification that all of the Rubisco amino acid residues modified by DEP at pH 6.0 were histidine was accomplished by simultaneous determination of (DELTA)A(,239) and {(\u2714)C}DEP-incorporation. We have demonstrated the presence of a single histidyl residue per 69-kilodalton enzyme protomer which appears to be essential for activity and resides in the large catalytic subunit by modification with DEP at pH 6.0. This histidyl residue may, indeed, function as the base required to create the carbanion at C-2 of RuBP in the proposed carboxylase reaction mechanism. Chemical modification by rose bengal, methylene blue, pyridoxal 5\u27-phosphate, potassium ferrate, cyanate and periodate were attempted, but problems with each experimental system allowed no definitive conclusions to be drawn regarding essential groups for catalysis or effector modulation in Rubisco. Characterization of homogeneous Rubisco isolated from diploid and tetraploid cultivars of perennial ryegrass reportedly differing in photorespiration revealed virtually identical K(,m) (CO(,2)) values for fully CO(,2)/Mg(\u272+)-activated enzyme and CO(,2)/Mg(\u272+)-activation and deactivation kinetics. The doubling of ploidy also had no effect on the quantity of total soluble protein or Rubisco protein in young, fully expanded ryegrass leaves. Hydroxylamine, a reported differential modulator of Rubisco, was found to inhibit both the carboxylase and oxygenase activities when assayed separately or simultaneously. Two inhibitory processes were observed: a slow reaction of hydroxylamine with RuBP and a rapid, direct inhibition of the enzyme