3-bromo-2-butanone 1,4-bisphosphate as an affinity label for ribulosebisphosphate carboxylase.
3-Bromo-2-butanone 1,4-bisphosphate has been synthesized in an attempt to find a reactive analog of ribulose 1,5-bisphosphate for labeling the active site of ribulosebisphosphate carboxylase (EC 4.1.1.39). The reagent irreversibly inactivates the carboxylase from spinach, and several observations suggest that the inactivation results from modification of an active-site residue: (1) Ribulose 1,5-bisphosphate protects against inactivation. (2) The extent of reagent incorporation shows that modification of one residue per catalytic site can account for the inactivation. (3) Comparisons of autoradiograms of peptide maps prepared from carboxylase treated with the (32)P-labeled reagent in the absence and presence of substrate indicate that inactivation results from a fairly selective modification. (4) Although the reagent's greatest inherent reactivity is toward sulfhydryl groups, inactivation of the enzyme is due to alteration of an amino-acid residue other than cysteine.
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830.
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Hartman
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Norton