Both carboxylase and oxygenase activities of ribulose- 1,5-bisphosphate (RuBP) carboxylase/oxygenase from a bacterial source, Rhodospirillum rubrum, and a plant source, spinach, were rapidly and completely inactivated by low concentrations of diethyl pyrocarbonate (DEP) at pH 7.0 and 30 °C. Inactivation kinetics were pseudo first order for up to 80% inactivation. The reaction order with respect to inactivation by DEP was approximately 1 for both enzymes, thereby indicating the modification of a single residue per subunit. One substrate, ribulose bisphosphate, the product 3-phosphoglycerate, and two competitive inhibitors, sedoheptulose 1,7-bisphosphate and 2-carboxyhexitol 1,6-bisphosphate, protected against inactivation, thereby indicating that DEP modifies the active site. DEP-modified enzyme showed an increase in the absorption at 240 nm, which was reversed upon treatment with hydroxylamine. The activity lost by DEP modification could be partially recovered after treatment with 0.5 M hydroxylamine at 25 °C for 2.5 h. The differential absorption at 240 nm suggests that DEP modified 4.5 and 2.2 histidine residues per large subunit of the enzymes from R. rubrum and spinach, respectively. After dissociation with sodium dodecyl sulfate of the spinach enzyme inactivated with [3H]DEP, polyacrylamide gel electrophoresis established the modification of large subunits only. Up to 65% loss of activity in the spinach enzyme was directly proportional to the number of histidine residues modified, and the extrapolation to 100% inactivation implied involvement of 1 histidine residue in activity per subunit. The differential absorption at 240 nm in the presence and absence of protectants coupled with differential labeling with [3H]DEP also indicated the modification of 1 essential histidine residue per subunit in RuBP carboxylase/ oxygenase from spinach and at least 1 per subunit for enzyme from R. rubrum.
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