Ribulose 1,5-bisphosphate carboxylase from spinach was rapidly inactivated by diethylpyrocarbonate (DEP) at pH 7.0 and 30°C. The inactivation showed saturation kinetics with a half-inactivation time at saturating DEP equal to 0.1 minutes and KDEP = 7.4 mM. One substrate, ribulose bisphosphate, the product 3-phosphoglycerate and two competitive inhibitors protected against inactivation, thereby indicating that DEP modifies the active site. DEP-modified enzyme showed an increased absorption at 240 nm which was lost upon treatment with 0.4 M hydroxylamine. Most of the activity lost by DEP modification could be restored after treatment with 0.4 M hydroxylamine at 4°C. The results suggest that DEP modified 2 to 3 histidine residues per 70,000-dalton combination of large and small subunits. These residues are essential to catalysis by the carboxylase activity of ribulose bisphosphate carboxylase/oxygenase.
|Original language||English (US)|
|Number of pages||7|
|Journal||Biochemical and Biophysical Research Communications|
|State||Published - Jun 30 1980|
Bibliographical noteFunding Information:
ACKNOWLEDGMENTS: We thank Dr. Fazal R. Khan during this work. This research was supported Herman Frasch Foundation and NIH (GM-19,972).