2-Bromoacetylaminopentitol 1,5-bisphosphate as an affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum
2-Bromoacetylaminopentitol 1,5-bisphosphate (BrAcNH-pentitol-P/sub 2/)(an epimeric mixture of 2-bromoacetylamino-2-deoxy-D-ribitol bisphosphate and 2-bromo-acetylamino-2-deoxy-D-arabinitol 1,5-bisphosphate) has been synthesized from D-ribulose 1,5-bisphosphate by reductive amination with sodium cyanoborohydride followed by bromoacetylation of the resultant amine with bromoacetyl bromide. Under conditions that favor full activation of the enzyme, ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is completely inactivated by BrAcNH-pentitol-P/sub 2/ in a pseudo-first order process. A rate saturation is observed with a minimal inactivation half-life of 38 min and K/sub inact/ for reagent of 0.38 mM. The competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduces the rate of inactivation, and kinetic analyses are consistent with the protection reflecting true competition of inhibitor and reagent for the same site. As shown with isotopically labeled reagent complete inactivation is associated with covalent incorporation of 1.1 mol of reagent/mol of subunit. Based on reversibility of inactivation by thiolysis and based on analysis of labeled products in acid hydrolysates of the modified enzyme, a methionyl sulfonium salt is the reaction product. In the absence of CO/sub 2/ and Mg/sup 2 +/ (ligands required for activation), the enzyme is resistant to BrAcNH-pentitol-P/sub 2/, which suggests that the site-specific modification of a methionyl residue requires a fully functional catalytic center.
- Research Organization:
- Oak Ridge National Lab., TN
- DOE Contract Number:
- W-7405-ENG-26
- OSTI ID:
- 6204554
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 257:7
- Country of Publication:
- United States
- Language:
- English
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