Inactivation of ribulosebisphosphate carboxylase/oxygenase from rhodospirillum rubrum and spinach with the new affinity label 2-bromo-1,5-dihydroxy-3-pentanone 1,5-bisphosphate |
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Authors: | Mark I Donnelly Fred C Hartman |
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Institution: | University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 USA |
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Abstract: | In an attempt to identify the active-site base believed to initiate catalysis by ribulosebisphosphate carboxylase, we have synthesized 2-bromo-1, 5-dihydroxy-3-pentanone 1,5-bisphosphate, a reactive analogue of a postulated intermediate of carboxylation. Although highly unstable, this compound can be shown to inactivate the carboxylases from both and spinach rapidly and irreversibly. Inactivation follows pseudo first-order kinetics, shows rate saturation and is greatly reduced by saturating amounts of the competitive inhibitor, 2-carboxyribitol 1,5-bisphosphate. The incorporation of reagent, quantified by reducing the modified carboxylases with 3H]NaBH4, shows that inactivation results from the modification of approximately one residue per catalytic subunit of the enzyme and less than one residue per protomeric unit of the spinach enzyme. |
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