Modification of histidine at the active site of spinach ribulose bisphosphate carboxylase

Ashok K. Saluja, Bruce A. McFadden

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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 languageEnglish (US)
Pages (from-to)1091-1097
Number of pages7
JournalBiochemical and Biophysical Research Communications
Issue number4
StatePublished - Jun 30 1980
Externally publishedYes

Bibliographical note

Funding Information:
ACKNOWLEDGMENTS: We thank Dr. Fazal R. Khan during this work. This research was supported Herman Frasch Foundation and NIH (GM-19,972).


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