Evidence for a reactive cysteine at the nucleotide binding site of spinach ribulose-5-phosphate kinase |
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| Authors: | J Omnaas M A Porter F C Hartman |
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| Affiliation: | 1. CNRS, UPS, F-310621 Toulouse, France;2. School of Mathematics and Statistics, Central South University, Changsha 410083, China;3. Department of Mathematics and Statistics, University of Wyoming, Laramie, WY 82070, USA;1. State Key Laboratory of Efficient Production of Forest Resources, Key Laboratory of Forest Management and Growth Modelling, National Forestry and Grassland Administration, Institute of Forest Resource Information Techniques, Chinese Academy of Forestry, Beijing 100091, PR China;2. Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing 100091, PR China;1. Department of Industrial Engineering and Management, Shanghai Jiao Tong University, Shanghai 200240, China;2. Delta-NTU Corporate Laboratory for Cyber-Physical System, School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798, Singapore;3. School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore 639798, Singapore;1. Faculty of Science and Engineering, International Campus-Kish Island, Sharif University of Technology (SUT), Iran;2. Department of Computer Science, ETH Zurich (ETHZ), Switzerland;3. Institute of Computer Engineering, Vienna University of Technology (TU Wien), Austria;4. Department of Computer Engineering, Sharif University of Technology (SUT), Iran;1. Université Clermont Auvergne, CNRS, CERDI, F-63000 Clermont-Ferrand, France;2. Department of Economics, Monash University, Australia;3. IFN, Sweden |
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| Abstract: | Ribulose-5-phosphate kinase from spinach was rapidly inactivated by N-bromoacetylethanolamine phosphate in a bimolecular fashion with a k2 of 2.0 M-1 S-1 at 2 degrees C and pH 8.0. Ribulose 5-phosphate had little effect on the rate of inactivation, whereas complete protection was afforded by ADP or ATP. The extent of incorporation as determined with 14C-labeled reagent was about 1 molar equivalent per subunit in the presence of ATP with full retention of enzymatic activity, and about 2 molar equivalents per subunit in the completely inactivated enzyme. Amino acid analyses of enzyme derivatized with 14C-labeled reagent reveal that all of the covalently incorporated reagent was associated with cysteinyl residues. Hence two sulfhydryls are reactive, but the inactivation correlates with alkylation of one cysteinyl residue at or near the enzyme's nucleotide binding site. The kinase was also extremely sensitive to the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethyl-maleimide. The reactive sulfhydryl groups are likely those generated by reduction of a disulfide during activation. |
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